Rna constructs and uses thereof

ABSTRACT

Disclosed herein are RNA polynucleotides comprising a 5′ Cap, a 5′ UTR comprising a cap proximal sequence disclosed herein, and a sequence encoding a payload. Also disclosed herein are compositions and medical preparations comprising the same, and methods of making and using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT Application No. PCT/EP2021/060508, filed Apr. 22, 2021, which claims foreign priority to International Patent Application Nos. PCT/EP2021/059947, filed on Apr. 16, 2021, PCT/EP2021/054622, filed on Feb. 24, 2021, PCT/EP2021/052716, filed on Feb. 4, 2021, PCT/EP2021/052572, filed on Feb. 3, 2021, PCT/EP2021/051772, filed on Jan. 26, 2021, PCT/EP2021/050874, filed on Jan. 15, 2021, PCT/EP2021/050875, filed on Jan. 15, 2021, PCT/EP2021/050027, filed on Jan. 4, 2021, PCT/EP2020/087844, filed on Dec. 23, 2020, PCT/EP2020/085653, filed on Dec. 10, 2020, PCT/EP2020/085145, filed on Dec. 8, 2020, PCT/EP2020/084342, filed on Dec. 2, 2020, PCT/EP2020/083435, filed on Nov. 25, 2020, PCT/EP2020/082989, filed on Nov. 20, 2020, PCT/EP2020/082601, filed on Nov. 18, 2020, PCT/EP2020/081981, filed on Nov. 12, 2020, PCT/EP2020/081544, filed on Nov. 9, 2020, PCT/EP2020/073668, filed on Aug. 24, 2020, PCT/EP2020/071839, filed on Aug. 3, 2020, PCT/EP2020/071733, filed on Jul. 31, 2020, PCT/EP2020/069805, filed on Jul. 13, 2020, PCT/EP2020/068174, filed on Jun. 26, 2020, PCT/EP2020/066968, filed on Jun. 18, 2020, PCT/EP2020/061239, filed on Apr. 22, 2020, the disclosures of each of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 31, 2022, is named 2013237-0219_SL.txt and is 152,913 bytes in size.

BACKGROUND

Use of RNA polynucleotides as therapeutics is a new and emerging field.

SUMMARY

The present disclosure identifies certain challenges that can be associated with RNA therapeutics.

For example, in some embodiments, the present disclosure identifies the source of certain problems that can be encountered with expression of polypeptides encoded by RNA therapeutics. Among other things, the present disclosure provides technologies for improving translation efficiency of an RNA encoding a payload, and/or expression of a polypeptide payload encoded by an RNA. In some embodiments, translation efficiency and/or expression of an RNA-encoded payload can be improved with an RNA polynucleotide comprising: a Cap1 structure (e.g., a m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG cap); a 5′ UTR comprising a cap proximal sequence disclosed herein, and a sequence encoding a payload. Without wishing to be bound by theory, the present disclosure proposes that improved translation efficiency and/or polypeptide payload expression can be achieved through preferential binding of eukaryotic translation initiation factor 4E (eIF4E), rather than IFN-induced protein with tetratricopeptide repeats-1 (IFIT1) to an RNA comprising a Cap1 structure, e.g., a m₂ ^(7′3′-O)Gppp(m₁ ^(2′-O))ApG cap, and/or a 5′ UTR comprising a cap proximal sequence disclosed herein. For example, in some embodiments, it is proposed that eIF4E may compete with IFIT1 for binding to an RNA polynucleotide based on a 5′ cap structure. Among other things, the present disclosure provides certain technologies that may favor eIF4E binding, at least relative to IFIT1 binding, and/or may otherwise enhance translation.

In some embodiments, the present disclosure teaches that identity of particular sequence(s) proximal to a 5′ cap (e.g., a 5′ Cap1 structure) can influence translation efficiency of an associated payload. Without wishing to be bound by any particular theory, the present disclosure proposes that eIF4E competes with IFIT1 for binding to an RNA polynucleotide based on the identity of one or more nucleotides downstream of a 5′ cap, e.g., a cap proximal sequence as disclosed herein. In some embodiments, the present disclosure demonstrates that an AGAAU or an AGCAC sequence downstream of a 5′ cap (e.g., a 5′ Cap 1 structure) can improve translation. The present disclosure proposes that presence of such sequence (e.g., AGAAU or an AGCAC can increase eIF4E binding, at least relative to IFIT1.

Alternatively or additionally, the present disclosure documents certain advantages of avoiding (e.g., ensuring that absence of) a self-hybridizing sequence (which may, in some instances, be referred to as a self-complementary sequence) in an RNA polynucleotide encoding a payload. For example, the present disclosure demonstrates that such absence can improve, and/or be required for, translation (e.g., translation efficiency) of an associated (e.g., RNA-encoded) payload, and/or otherwise for expression of a polypeptide encoded thereby. Without wishing to be bound by theory, it is believed that a self-hybridizing sequence (and in particular a sequence that hybridizes with sequences within or comprising one or more of a Kozak sequence, a 5′ UTR element, and/or a 3′ UTR element) interfere with one or more aspects of translation. For example, in some embodiments, it is proposed that such self-hybridization may inhibit binding of transcription and/or translation factors to an RNA polynucleotide by self-hybridizing to a complementary sequence in said RNA polynucleotide.

Still further alternatively or additionally, in some embodiments, the present disclosure defines particular lipid components, and/or ratios thereof, that may be especially useful or effective in delivering nucleic acids, and in particular RNAs (e.g., therapeutic RNAs or other RNAs encoding a polypeptide) upon administration (e.g., by injection, such as by intramuscular injection or by intravenous injection) to a subject. For example, in some embodiments, the present disclosure demonstrates that lipid ALC-0315 is unusually and particularly useful for delivery as described herein.

Disclosed herein, inter alia, is a composition or medical preparation comprising an RNA polynucleotide, comprising: (i) a 5′ cap that is or comprises a cap1 structure, e.g., as disclosed herein; (ii) a 5′ UTR sequence comprising a cap proximal sequence, e.g., as disclosed herein; and (iii) a sequence encoding a payload. Also disclosed herein are methods of making and using the same to, e.g., induce an immune response in a subject.

Provided herein is a composition or medical preparation comprising an RNA polynucleotide comprising:

a 5′ cap comprising a Cap1 structure; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein:

-   -   (i) the Cap1 structure comprises m7G(5′)ppp(5′)(2′OMeN₁)pN₂,         wherein N₁ is position +1 of the RNA polynucleotide, and N2 is         position +2 of the RNA polynucleotide, and wherein N₁ and N₂ are         each independently chosen from: A, C, G, or U; and     -   (ii) the cap proximal sequence comprises N₁ and N₂ of the Cap1         structure, and:         -   (a) a sequence selected from the group consisting of: A₃A₄X₅             (SEQ ID NO: 1); C₃A₄X₅ (SEQ ID NO: 2); A₃C₄A₅ (SEQ ID NO: 3)             and A₃U₄G₅ (SEQ ID NO: 4); or         -   (b) a sequence comprising: X₃Y₄X₅ (SEQ ID NO: 7);         -   wherein X₃ (nucleotide X at position +3 in SEQ ID NO: 7) or             X₅ (nucleotide X at position +5 in SEQ ID NO: 1 or SEQ ID             NO: 2) is each independently chosen from A, G, C, or U; and         -   wherein Y4 (nucleotide Y at position +4 in SEQ ID NO: 7) is             not C.

This disclosure also provides a composition or medical preparation comprising an RNA polynucleotide comprising: a 5′ cap; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of an RNA polynucleotide; and a sequence encoding a payload, wherein:

-   -   (i) the 5′ cap comprises a Cap1 structure comprising G*ppp(m₁         ^(2-O))N₁pN₂, wherein:         -   N1 is position +1 of the RNA polynucleotide, and N₂ is             position +2 of the RNA polynucleotide, and wherein N₁ and N₂             are each independently chosen from: A, C, G, or U; and         -   G* comprises the following structure:

-   -   -   wherein             represents the bond by which G* is bound to the first             phosphor atom         -   of the ppp group, R¹ is CH3, R² is OH or O—CH3, and R³ is             O—CH3; and

    -   (ii) the cap proximal sequence comprises N₁ and N₂ of the Cap1         structure, and:         -   (a) a sequence selected from the group consisting of: A₃A₄X₅             (SEQ ID NO: 1); C₃A₄X₅ (SEQ ID NO: 2); A₃C₄A₅ (SEQ ID NO: 3)             and A₃U₄G₅ (SEQ ID NO: 4); or         -   (b) a sequence comprising: X₃Y4X₅ (SEQ ID NO: 7);         -   wherein X₃ (nucleotide X at position +3 in SEQ ID NO: 7) or             X₅ (nucleotide X at position +5 in SEQ ID NO: 1 or SEQ ID             NO: 2) is each independently chosen from A, G, C, or U; and         -   wherein Y₄ (nucleotide Y at position +4 in SEQ ID NO: 7) is             not C.

Also provided herein is a composition or medical preparation comprising an RNA polynucleotide comprising:

a 5′ cap comprising a Cap1 structure; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein:

-   -   (i) the Cap1 structure comprises         m7(3′OMeG)(5′)ppp(5′)(2′OMeA₁)pG₂, wherein A₁ is position +1 of         the RNA polynucleotide, and G₂ is position +2 of the RNA         polynucleotide; and     -   (ii) the cap proximal sequence comprises A₁ and G₂ of the Cap1         structure, and a sequence comprising: A₃A₄U₅ (SEQ ID NO: 5) at         positions +3, +4 and +5 respectively of the RNA polynucleotide.

This disclosure provides a composition or medical preparation comprising a capped RNA polynucleotide encoding a gene product, which RNA polynucleotide comprises the formula:

wherein R¹ is CH3, R² is OH or O—CH3, and R³ is O—CH3, wherein B₁ is any nucleobase, preferably A; B2 is any nucleobase, preferably G; B₃ is any nucleobase, preferably A or C; B4 is any nucleobase; and B₅ is any nucleobase, and wherein, when the RNA polynucleotide is administered to a subject, the levels of expression of the encoded gene product at about 6 hours after administration and at about 48 hours after administration do not differ by more than 5-fold.

Provided herein is a pharmaceutical composition comprising an RNA polynucleotide disclosed herein. In some embodiments, a pharmaceutical composition comprises a composition or a medical preparation disclosed herein.

Also provided herein is a method of manufacturing a pharmaceutical composition, e.g., comprising an RNA polynucleotide disclosed herein, by combining an RNA polynucleotide with lipids to form lipid nanoparticles that encapsulate said RNA.

This disclosure provides a nucleic acid template suitable to produce a cap1-capped RNA, in which the first five nucleotides transcribed from the template strand of the nucleic acid template comprise the sequence N₁pN₂pN₃pN₄pN₅, wherein N₁ is any nucleotide, preferably T; N₂ is any nucleotide, preferably C; N₃ is any nucleotide, preferably T or G; N₄ is any nucleotide; and N₅ is any nucleotide. In some embodiments, a DNA template comprises: a 5′ UTR, a sequence encoding a payload, a 3′ UTR and a polyA sequence.

Provided herein is an vitro transcription reaction comprising:

(i) a template DNA comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence disclosed herein;

(ii) a polymerase; and

(iii) an RNA polynucleotide.

Also provided herein is an RNA polynucleotide isolated from an in vitro transcription reaction provided.

This disclosure provides, a method for producing a capped RNA comprising, transcribing a nucleic acid template in the presence of a cap structure, wherein the cap structure comprises G*ppp(m₁ ^(2′-O))N₁pN₂,

wherein N₁ is complementary to position +1 of the nucleic acid template and N₂ is complementary to position +2 of the nucleic acid template, and N₁ and N₂ are independently chosen from A, C, G or U, wherein position +3 of the nucleic acid template is any nucleotide, preferably T or G; position +4 of the nucleic acid template is any nucleotide; and position +5 of the nucleic acid template is any nucleotide, wherein G* comprises the following structure:

wherein

represents the bond by which G* is bound to the first phosphor atom of the ppp group, R¹ is CH₃, R² is OH or O—CH₃, and R³ is O—CH₃.

Also provided herein is a composition comprising a DNA polynucleotide comprising a sequence complementary to an RNA polynucleotide sequence provided. In some embodiments, a DNA polynucleotide disclosed herein can be used to transcribe an RNA polynucleotide disclosed herein.

This disclosure provides, a method comprising: administering to a subject, a pharmaceutical composition comprising an RNA polynucleotide disclosed herein formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle, e.g., as disclosed herein.

Also provided herein is a method of inducing an immune response in a subject, comprising administering to a subject, a pharmaceutical composition comprising an RNA polynucleotide disclosed herein formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle, e.g., as disclosed herein

Provided herein is a method of vaccination of a subject by administering a pharmaceutical composition comprising an RNA polynucleotide disclosed herein formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle, e.g., as disclosed herein.

This disclosure provides, a method of decreasing interaction with IFIT1 of an RNA polynucleotide that comprises a 5′ cap and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, the method comprising a step of:

providing a variant of an RNA polynucleotide that differs from a parental RNA polynucleotide by substitution of one or more residues within a cap proximal sequence, and

determining that interaction of a variant with IFIT1 is decreased relative to that of a parental RNA polynucleotide.

Also provided herein is a method of producing a polypeptide comprising a step of:

providing an RNA polynucleotide that comprises a 5′ cap, a cap proximal sequence that comprises positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, and a sequence encoding a payload;

wherein an RNA polynucleotide is characterized in that when assessed in an organism administered an RNA polynucleotide or a composition comprising the same, elevated expression and/or increased duration of expression of a payload is observed relative to an appropriate reference comparator.

Provided herein is a method of increasing translatability of an RNA polynucleotide that comprises a 5′ cap, a cap proximal sequence that comprises positions +1, +2, +3, +4, and +5 of the RNA polynucleotide and a sequence encoding a payload, the method comprising a step of:

providing a variant of an RNA polynucleotide that differs from a parental RNA polynucleotide by substitution of one or more residues within a cap proximal sequence; and

determining that expression of a variant is increased relative to that of a parental RNA polynucleotide.

Provided herein is a therapeutic RNA comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises: including one or more of the following residues in a cap proximal sequence: X at position +1 of an RNA polynucleotide, X at position +2 of an RNA polynucleotide, A at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and X at position +5 of an RNA polynucleotide, demonstrated to increase expression of an RNA when administered to a subject in an LNP formulation. In some embodiments, X is chosen from A, C, G or U.

This disclosed provides, a therapeutic RNA comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises: including one or more of the following residues in a cap proximal sequence: X at position +1 of an RNA polynucleotide, X at position +2 of an RNA polynucleotide, C at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and X at position +5 of an RNA polynucleotide, demonstrated to increase expression of an RNA when administered to a subject in an LNP formulation. In some embodiments, X is chosen from A, C, G or U.

Also provided herein is therapeutic RNA comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises:including one or more of the following residues in a cap proximal sequence: A at position +1 of an RNA polynucleotide, G at position +2 of an RNA polynucleotide, A at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and U at position +5 of an RNA polynucleotide, demonstrated to increase expression of an RNA when administered to a subject in an LNP formulation.

This disclosure provides, a method of increasing translation of an RNA polynucleotide comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises: including one or more of the following residues in a cap proximal sequence: X at position +1 of an RNA polynucleotide, X at position +2 of an RNA polynucleotide, A at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and X at position +5 of an RNA polynucleotide. In some embodiments, X is chosen from A, C, G or U.

Provided herein is a method of increasing translation of an RNA polynucleotide comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises: including one or more of the following residues in a cap proximal sequence: X at position +1 of an RNA polynucleotide, X at position +2 of an RNA polynucleotide, C at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and X at position +5 of an RNA polynucleotide. In some embodiments, X is chosen from A, C, G or U.

Also provided herein is a method of increasing translation of an RNA polynucleotide comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises: including one or more of the following residues in a cap proximal sequence: A at position +1 of an RNA polynucleotide, G at position +2 of an RNA polynucleotide, A at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and U at position +5 of an RNA polynucleotide.

Also provided herein is a method of providing a framework for an RNA polynucleotide that comprises a 5′ cap, a cap proximal sequence, and a payload sequence, the method comprising a step of:

assessing at least two variants of an RNA polynucleotide, wherein:

each variant includes a same 5′ cap and payload sequence; and

the variants differ from one another at one or more specific residues of a cap proximal sequence;

wherein the assessing comprises determining expression levels and/or duration of expression of a payload sequence; and

selecting at least one combination of 5′ cap and a cap proximal sequence that displays elevated expression relative to at least one other combination.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 demonstrates plasma levels of EPO at 6, 24, 48 and 72 hours after intravenous administration of mice with murine EPO (mEPO) mRNA constructs with or without a Lig3 presence in the 3′UTR sequence. Blood was collected 6, 24, 48 and 72 hours after administration and samples were analyzed for mEPO levels via ELISA.

FIGS. 2A-2C are schematics of a Lig3 sequence self-hybridizing to a 5′ UTR (FIG. 2A-2B) or a 3′ UTR (FIG. 2C). FIG. 2A discloses SEQ ID NOs: 43-44, respectively, in order of appearance. FIG. 2B discloses SEQ ID NOs: 45 and 44, respectively, in order of appearance. FIG. 2C discloses SEQ ID NOs: 46-47, respectively, in order of appearance.

FIGS. 3A-3I are structures of 5′ caps that can be incorporated into mRNAs.

FIG. 4 demonstrates plasma levels of mEPO at 6, 24, 48 and 72 hours after intravenous administration of mice with mEPO mRNA constructs having different nucleotides at the +3, +4 or +5 positions. Blood was collected 6, 24, 48 and 72 hours after administration and samples were analyzed for mEPO levels via ELISA. FIG. 4 discloses SEQ ID NOs: 5, 3, 4 and 6 respectively, in order of appearance.

FIG. 5 demonstrates an anti-S protein IgG response 7, 14, 21 and 28 d after immunization with BNT162a1. BALB/c mice were immunized IM once with 1, 5 or 10 μg of LNP-formulated RBL063.3. On day 7, 14, 21 and 28 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. For day 7, day 14, day 21 and day 28, values for a serum dilution of 1:100 were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

FIG. 6 demonstrates Anti-S protein IgG response 7, 14, 21 and 28 d after immunization with BNT162b1. BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulated RBP020.3. On day 7, 14, 21 and 28 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. For day 7 (1:100), day 14 (1:300), day 21 (1:900), and day 28 (1:2700) different serum dilutions were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

FIG. 7 demonstrates neutralization of SARS-CoV-2 pseudovirus 14, 21 and 28 d after immunization with BNT162b1. BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulated RBP020.3. On 14, 21 and 28 d after immunization, animals were bled, and the sera were tested for SARS-CoV-2 pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean+SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification.

FIG. 8 demonstrates Anti-S protein IgG response 7, 14 and 21 d after immunization with BNT162c1. BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulated RBS004.3. On day 7, 14 and 21 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. For day 7 (1:100), day 14 (1:300), and day 21 (1:900) different serum dilution were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

FIG. 9 demonstrates neutralization of SARS-CoV-2 pseudovirus 14 and 21 d after immunization with BNT162c1. BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulated RBS004.3. On 14 and 21 d after immunization, animals were bled and the sera were tested for SARS-CoV-2 pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean+SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification.

FIG. 10 demonstrates anti-S protein IgG response 7, 14, 21 and 28 d after immunization with LNP-formulated RBL063.1. BALB/c mice were immunized IM once with 1, 5 or 10 μg of LNP-formulated RBL063.1. On day 7, 14, 21 and 28 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. For day 7 (1:100), day 14 (1:100), day 21 (1:300) and day 28 (1:900) different serum dilution were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

FIG. 11 shows neutralization of SARS-CoV-2 pseudovirus 14, 21 and 28 d after immunization with LNP-formulated RBL063.1. BALB/c mice were immunized IM once with 1, 5 or 10 μg of LNP-formulated RBL063.1. On 14, 21, and 28 d after immunization, animals were bled and the sera were tested for SARS-CoV-2 pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean+SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification.

FIG. 12 shows Anti-S protein IgG response 7, 14 and 21 d after immunization with BNT162b2 (LNP-formulated RBP020.1). BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulated RBP020.1. On day 7, 14, and 21 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. For day 7 (1:100), day 14 (1:300), and day 21 (1:1100) different serum dilution were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

FIG. 13 demonstrates neutralization of SARS-CoV-2 pseudovirus 14 and 21 after immunization with BNT162b2 (LNP-formulated RBP020.1). BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulated RBP020.1. On day 14 and 21 after immunization, animals were bled and the sera were tested for SARS-CoV-2 pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean+SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification.

FIG. 14 shows anti-S protein IgG response 7, 14 and 21 d after immunization with LNP-formulated RBS004.2. BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulated RBS004.2. On day 7, 14 and 21 after immunization, animals were bled and the serum samples were analyzed for total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) measured via ELISA. For day 7 (1:100), day 14 (1:300), and day 21 (1:900) different serum dilution were included in the graph. One point in the graph stands for one mouse, every mouse sample was measured in duplicates (group size n=8; mean+SEM is included for the groups).

FIG. 15 demonstrates neutralization of SARS-CoV-2 pseudovirus 14 and 21 after immunization with LNP-formulated RBS004.2. BALB/c mice were immunized IM once with 0.2, 1 or 5 μg of LNP-formulated RBS004.2. On 14, and 21 d after immunization, animals were bled, and the sera were tested for SARS-CoV-2 pseudovirus neutralization. Graphs depict pVN50 serum dilutions (50% reduction of infectious events, compared to positive controls without serum). One point in the graphs stands for one mouse. Every mouse sample was measured in duplicate. Group size n=8. Mean+SEM is shown by horizontal bars with whiskers for each group. LLOQ, lower limit of quantification. ULOQ, upper limit of quantification.

FIG. 16 depicts ALC-0315 activity in the screening process.

FIGS. 17A-17B demonstrate luciferase expression was monitored on (FIG. 17A) the ventral (drainage to the liver) and (FIG. 17B) the right (site of injection) and dorsal (site of injection) sides of the animal after intramuscular administration in wild-type (WT) or ApoE knockout C57B1/6 mice in the presence or absence of ApoE3. Luciferase expression was detected using Xenolight D-Luciferin Rediject at 4, 24, 72 and 96 hours post administration.

FIG. 18 shows luciferase activity after intravenous (IV) and intramuscular (IM) administration in wild-type (WT) or ApoE knockout C57B1/6 mice in the presence (KO+) or absence (KO) of ApoE3. Luciferase expression was detected using Xenolight D-Luciferin Rediject at 4 hours post administration.

CERTAIN DEFINITIONS

Although the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodologies, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

In the following, the elements of the present disclosure will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to disclose and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutations and combinations of all described elements should be considered disclosed by this description unless the context indicates otherwise. The term “about” means approximately or nearly, and in the context of a numerical value or range set forth herein in some embodiments means±20%, +10%, +5%, or +3% of the numerical value or range recited or claimed.

The terms “a” and “an” and “the” and similar reference used in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it was individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”), provided herein is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

Unless expressly specified otherwise, the term “comprising” is used in the context of the present document to indicate that further members may optionally be present in addition to the members of the list introduced by “comprising”. It is, however, contemplated as a specific embodiment of the present disclosure that the term “comprising” encompasses the possibility of no further members being present, i.e., for the purpose of this embodiment “comprising” is to be understood as having the meaning of “consisting of” or “consisting essentially of”.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present disclosure was not entitled to antedate such disclosure.

In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

Agent: As used herein, the term “agent”, may refer to a physical entity or phenomenon. In some embodiments, an agent may be characterized by a particular feature and/or effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety.

Amino acid: in its broadest sense, as used herein, the term “amino acid” refers to a compound and/or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

Analog: As used herein, the term “analog” refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.

Antibody agent: As used herein, the term “antibody agent” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. For example, in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent in or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art to correspond to CDRs1, 2, and 3 of an antibody variable domain; in some such embodiments, an antibody agent in or comprises a polypeptide or set of polypeptides whose amino acid sequence(s) together include structural elements recognized by those skilled in the art to correspond to both heavy chain and light chain variable region CDRs, e.g., heavy chain CDRs 1, 2, and/or 3 and light chain CDRs 1, 2, and/or 3. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, an antibody agent may be or comprise a polyclonal antibody preparation. In some embodiments, an antibody agent may be or comprise a monoclonal antibody preparation. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a particular organism, such as a camel, human, mouse, primate, rabbit, rat; in many embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a human. In some embodiments, an antibody agent may include one or more sequence elements that would be recognized by one skilled in the art as a humanized sequence, a primatized sequence, a chimeric sequence, etc. In some embodiments, an antibody agent may be a canonical antibody (e.g., may comprise two heavy chains and two light chains). In some embodiments, an antibody agent may be in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi-specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab′ fragments, F(ab′)2 fragments, Fd′ fragments, Fd fragments, and isolated CDRs or sets thereof, single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.].

Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of, susceptibility to, severity of, stage of, etc the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

Binding: It will be understood that the term “binding”, as used herein, typically refers to a non-covalent association between or among two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts—including where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and/or in a biological system or cell). Binding between two entities may be considered “specific” if, under the conditions assessed, the relevant entities are more likely to associate with one another than with other available binding partners.

Biological Sample: As used herein, the term “biological sample” typically refers to a sample obtained or derived from a biological source (e.g., a tissue or organism or cell culture) of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.

Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

Corresponding to: As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” may be used to designate the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid “corresponding to” a residue at position 190, for example, need not actually be the_(190t)h amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and/or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term “corresponding to” may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as “corresponding to” a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and/or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.

Designed: As used herein, the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and/or (iii) that is distinct from natural substances and other known agents.

Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.

Epitope: as used herein, the term “epitope” refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).

Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of any gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.

Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation.

Polypeptide: As used herein refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.

Prevent or prevention: as used herein when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.

Risk: as will be understood from context, “risk” of a disease, disorder, and/or condition refers to a likelihood that a particular individual will develop the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and/or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes.

Susceptible to: An individual who is “susceptible to” a disease, disorder, and/or condition is one who has a higher risk of developing the disease, disorder, and/or condition than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

Vaccination: As used herein, the term “vaccination” refers to the administration of a composition intended to generate an immune response, for example to a disease-associated (e.g., disease-causing) agent. In some embodiments, vaccination can be administered before, during, and/or after exposure to a disease-associated agent, and in certain embodiments, before, during, and/or shortly after exposure to the agent. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of a vaccine composition. In some embodiments, vaccination generates an immune response to an infectious agent. In some embodiments, vaccination generates an immune response to a tumor; in some such embodiments, vaccination is “personalized” in that it is partly or wholly directed to epitope(s) (e.g., which may be or include one or more neoepitopes) determined to be present in a particular individual's tumors.

Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present disclosure provides, among other things, an RNA polynucleotide comprising (i) a 5′ cap that is or comprises a cap1 structure, e.g., as disclosed herein; (ii) a 5′ UTR sequence comprising a cap proximal sequence, e.g., as disclosed herein; and (iii) a sequence encoding a payload. Also provided herein are compositions and medical preparations comprising the same, as well as methods of making and using the same. In some embodiments, translation efficiency of an RNA encoding a payload, and/or expression of a payload encoded by an RNA, can be improved with an RNA polynucleotide comprising a 5′ cap comprising a Cap1 structure disclosed herein, e.g., m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG cap; a 5′ UTR comprising a cap proximal sequence disclosed herein, and a sequence encoding a payload. In some embodiments, absence of a self-hybridizing sequence in an RNA polynucleotide encoding a payload can further improve translation efficiency of an RNA encoding a payload, and/or expression of a payload encoded by an RNA payload.

RNA Polynucleotide

The term “polynucleotide” or “nucleic acid”, as used herein, refers to DNA and RNA such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. A nucleic acid may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the invention, a polynucleotide is preferably isolated.

In some embodiments, nucleic acids may be comprised in a vector. The term “vector” as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). In some embodiments, a vector may be an expression vector; alternatively or additionally, in some embodiments, a vector may be a cloning vector. Those skilled in the art will appreciate that, in some embodiments, an expression vector may be, for example, a plasmid; alternatively or additionally, in some embodiments, an expression vector may be a viral vector. Typically, an expression vector will contain a desired coding sequence and appropriate other sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired fragment (typically a DNA fragment), and may lack functional sequences needed for expression of the desired fragment(s).

In some embodiments, a nucleic acid as described and/or utilized herein may be or comprise recombinant and/or isolated molecules.

Those skilled in the art, reading the present disclosure, will understand that the term “RNA” typically refers to a nucleic acid molecule which includes ribonucleotide residues. In some embodiments, an RNA contains all or a majority of ribonucleotide residues. As used herein, “ribonucleotide” refers to a nucleotide with a hydroxyl group at the 2′-position of a 3-D-ribofuranosyl group. In some embodiments, an RNA may be partly or fully double stranded RNA; in some embodiments, an RNA may comprise two or more distinct nucleic acid strands (e.g., separate molecules) that are partly or fully hybridized with one another. In many embodiments, an RNA is a single strand, which may in some embodiments, self-hybridize or otherwise fold into secondary and/or tertiary structures. In some embodiments, an RNA as described and/or utilized herein does not self-hybridize, at least with respect to certain sequences as described herein. In some embodiments, an RNA may be an isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and/or a modified RNA (where the term “modified” is understood to indicate that one or more residues or other structural elements of the RNA differs from naturally occurring RNA; for example, in some embodiments, a modified RNA differs by the addition, deletion, substitution and/or alteration of one or more nucleotides and/or by one or more moieties or characteristics of a nucleotide—e.g., of a nucleoside or of a backbone structure or linkage). In some embodiments, a modification may be or comprise addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA (e.g., in a modified RNA) may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs are considered analogs of naturally-occurring RNA.

In some embodiments of the present disclosure, an RNA is or comprises messenger RNA (mRNA) that relates to an RNA transcript which encodes a polypeptide.

In some embodiments, an RNA disclosed herein comprises: a 5′ cap comprising a 5′ cap disclosed herein; a 5′ untranslated region comprising a cap proximal sequence (5′-UTR), a sequence encoding a payload (e.g., a polypeptide); a 3′ untranslated region (3′-UTR); and/or a polyadenylate (PolyA) sequence.

In some embodiments, an RNA disclosed herein comprises the following components in 5′ to 3′ orientation: a 5′ cap comprising a 5′ cap disclosed herein; a 5′ untranslated region comprising a cap proximal sequence (5′-UTR), a sequence encoding a payload (e.g., a polypeptide); a 3′ untranslated region (3′-UTR); and a PolyA sequence.

In some embodiments, an RNA is produced by in vitro transcription or chemical synthesis. In some embodiments, an mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides.

In some embodiments, an RNA disclosed herein is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

In some embodiments, an RNA is “replicon RNA” or simply a “replicon”, in particular “self-replicating RNA” or “self-amplifying RNA”. In some embodiments, a replicon or self-replicating RNA is derived from or comprises elements derived from a ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5′-cap, and a 3′ poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsP1-nsP4) are typically encoded together by a first ORF beginning near the 5′ terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3′ terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2:1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA polynucleotide that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234). Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.

In some embodiments, an RNA described herein may have modified nucleosides. In some embodiments, an RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.

The term “uracil,” as used herein, describes one of the nucleobases that can occur in the nucleic acid of RNA. The structure of uracil is:

The term “uridine,” as used herein, describes one of the nucleosides that can occur in RNA. The structure of uridine is:

UTP (uridine 5′-triphosphate) has the following structure:

Pseudo-UTP (pseudouridine-5′-triphosphate) has the following structure:

“Pseudouridine” is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond. Another exemplary modified nucleoside is N1-methylpseudouridine (m1Ψ), which has the structure:

N1-methylpseudouridine-5′-triphosphate (m1ΨTP) has the following structure:

Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the structure:

In some embodiments, one or more uridine in an RNA described herein is replaced by a modified nucleoside. In some embodiments, a modified nucleoside is a modified uridine. In some embodiments, an RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, an RNA comprises a modified nucleoside in place of each uridine.

In some embodiments, a modified nucleoside is independently selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U). In some embodiments, a modified nucleoside comprises pseudouridine (Ψ). In some embodiments, a modified nucleoside comprises N1-methyl-pseudouridine (m1Ψ). In some embodiments, a modified nucleoside comprises 5-methyluridine (m5U). In some embodiments, an RNA may comprise more than one type of modified nucleoside, and a modified nucleosides are independently selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U). In some embodiments, a modified nucleosides comprise pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ). In some embodiments, a modified nucleosides comprise pseudouridine (Ψ) and 5-methyluridine (m5U). In some embodiments, a modified nucleosides comprise N1-methylpseudouridine (m1Ψ) and 5-methyluridine (m5U). In some embodiments, a modified nucleosides comprise pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U).

In some embodiments, a modified nucleoside replacing one or more, e.g., all, uridine in the RNA may be any one or more of 3-methyl-uridine (m³U), 5-methoxy-uridine (mo⁵U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U), 4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm's²U), 5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine (mnm⁵U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U), 5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine (cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp³U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³Ψ), 5-(isopentenylaminomethyl)uridine (inm⁵U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um), 2′-O-methyl-pseudouridine (Ψm), 2-thio-2′-O-methyl-uridine (s²Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um), 3,2′-O-dimethyl-uridine (m³Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

In some embodiments, an RNA comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine. For example, in some embodiments, in an RNA 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine. In some embodiments, an RNA comprises 5-methylcytidine and one or more selected from pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), and 5-methyl-uridine (m5U). In some embodiments, an RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1Ψ). In some embodiments, the RNA comprises 5-methylcytidine in place of each cytidine and N1-methyl-pseudouridine (m1Ψ) in place of each uridine.

In some embodiments, an RNA encoding a payload, e.g., a vaccine antigen, is expressed in cells of a subject treated to provide a payload, e.g., vaccine antigen. In some embodiments, the RNA is transiently expressed in cells of the subject. In some embodiments, the RNA is in vitro transcribed RNA. In some embodiments, expression of a payload, e.g., a vaccine antigen is at the cell surface. In some embodiments, a payload, e.g., a vaccine antigen is expressed and presented in the context of MHC. In some embodiments, expression of a payload, e.g., a vaccine antigen is into the extracellular space, i.e., the vaccine antigen is secreted.

In the context of the present disclosure, the term “transcription” relates to a process, wherein the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA may be translated into peptide or protein.

According to the present invention, the term “transcription” comprises “in vitro transcription”, wherein the term “in vitro transcription” relates to a process wherein RNA, in particular mRNA, is in vitro synthesized in a cell-free system, preferably using appropriate cell extracts. Preferably, cloning vectors are applied for the generation of transcripts. These cloning vectors are generally designated as transcription vectors and are according to the present invention encompassed by the term “vector”. According to the present invention, the RNA used in the present invention preferably is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription according to the invention is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

With respect to RNA, the term “expression” or “translation” relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

In some embodiments, after administration of an RNA described herein, e.g., formulated as RNA lipid particles, at least a portion of the RNA is delivered to a target cell. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the peptide or protein it encodes. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell or macrophage. RNA particles such as RNA lipid particles described herein may be used for delivering RNA to such target cell. Accordingly, the present disclosure also relates to a method for delivering RNA to a target cell in a subject comprising the administration of the RNA particles described herein to the subject. In some embodiments, the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the peptide or protein encoded by the RNA. “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

In some embodiments, nucleic acid compositions described herein, e.g., compositions comprising a lipid nanoparticle encapsulated mRNA are characterized by (e.g., when administered to a subject) sustained expression of an encoded polypeptide. For example, in some embodiments, such compositions are characterized in that, when administered to a human, they achieve detectable polypeptide expression in a biological sample (e.g., serum) from such human and, in some embodiments, such expression persists for a period of time that is at least at least 36 hours or longer, including, e.g., at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours, or longer.

In some embodiments, the RNA encoding vaccine antigen to be administered according to the invention is non-immunogenic. RNA encoding immunostimulant may be administered according to the invention to provide an adjuvant effect. The RNA encoding immunostimulant may be standard RNA or non-immunogenic RNA.

The term “non-immunogenic RNA” as used herein refers to RNA that does not induce a response by the immune system upon administration, e.g., to a mammal, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the non-immunogenic RNA non-immunogenic, i.e., than would have been induced by standard RNA (stdRNA). In one preferred embodiment, non-immunogenic RNA, which is also termed modified RNA (modRNA) herein, is rendered non-immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and removing double-stranded RNA (dsRNA).

For rendering the non-immunogenic RNA non-immunogenic by the incorporation of modified nucleosides, any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleosides comprises a replacement of one or more uridines with a nucleoside comprising a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl-uridine (m³U), 5-methoxy-uridine (mo⁵U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U), 4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo⁵U), uridine 5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U), 5-methoxycarbonylmethyl-uridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U), 5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine (mnm⁵U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U), 5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U), 5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine (cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm5s²U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp³U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³Ψ), 5-(isopentenylaminomethyl)uridine (inm⁵U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um), 2′-O-methyl-pseudouridine (Ψm), 2-thio-2′-O-methyl-uridine (s²Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um), 3,2′-O-dimethyl-uridine (m³Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine. In one particularly preferred embodiment, the nucleoside comprising a modified nucleobase is pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ) or 5-methyl-uridine (m5U), in particular N1-methyl-pseudouridine.

In some embodiments, the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines. During synthesis of mRNA by in vitro transcription (IVT) using T7 RNA polymerase significant amounts of aberrant products, including double-stranded RNA (dsRNA) are produced due to unconventional activity of the enzyme. dsRNA induces inflammatory cytokines and activates effector enzymes leading to protein synthesis inhibition. dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reversed phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzymatic based method using E. coli RNaseIII that specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations can be used. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material.

As the term is used herein, “remove” or “removal” refers to the characteristic of a population of first substances, such as non-immunogenic RNA, being separated from the proximity of a population of second substances, such as dsRNA, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance. However, a population of first substances characterized by the removal of a population of second substances has a measurably lower content of second substances as compared to the non-separated mixture of first and second substances.

In some embodiments, the removal of dsRNA from non-immunogenic RNA comprises a removal of dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, the non-immunogenic RNA is free or essentially free of dsRNA. In some embodiments, the non-immunogenic RNA composition comprises a purified preparation of single-stranded nucleoside modified RNA. For example, in some embodiments, the purified preparation of single-stranded nucleoside modified RNA is substantially free of double stranded RNA (dsRNA). In some embodiments, the purified preparation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

In some embodiments, the non-immunogenic RNA is translated in a cell more efficiently than standard RNA with the same sequence. In some embodiments, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In some embodiments, translation is enhanced by a 3-fold factor. In some embodiments, translation is enhanced by a 4-fold factor. In some embodiments, translation is enhanced by a 5-fold factor. In some embodiments, translation is enhanced by a 6-fold factor. In some embodiments, translation is enhanced by a 7-fold factor. In some embodiments, translation is enhanced by an 8-fold factor. In some embodiments, translation is enhanced by a 9-fold factor. In some embodiments, translation is enhanced by a 10-fold factor. In some embodiments, translation is enhanced by a 15-fold factor. In some embodiments, translation is enhanced by a 20-fold factor. In some embodiments, translation is enhanced by a 50-fold factor. In some embodiments, translation is enhanced by a 100-fold factor. In some embodiments, translation is enhanced by a 200-fold factor. In some embodiments, translation is enhanced by a 500-fold factor. In some embodiments, translation is enhanced by a 1000-fold factor. In some embodiments, translation is enhanced by a 2000-fold factor. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-100-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold. In some embodiments, the factor is 10-500-fold. In some embodiments, the factor is 20-1000-fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000-fold. In some embodiments, the factor is 100-1000-fold. In some embodiments, the factor is 200-1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.

In some embodiments, the non-immunogenic RNA exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In some embodiments, the non-immunogenic RNA exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by a 3-fold factor. In some embodiments, innate immunogenicity is reduced by a 4-fold factor. In some embodiments, innate immunogenicity is reduced by a 5-fold factor. In some embodiments, innate immunogenicity is reduced by a 6-fold factor. In some embodiments, innate immunogenicity is reduced by a 7-fold factor. In some embodiments, innate immunogenicity is reduced by a 8-fold factor. In some embodiments, innate immunogenicity is reduced by a 9-fold factor. In some embodiments, innate immunogenicity is reduced by a 10-fold factor. In some embodiments, innate immunogenicity is reduced by a 15-fold factor. In some embodiments, innate immunogenicity is reduced by a 20-fold factor. In some embodiments, innate immunogenicity is reduced by a 50-fold factor. In some embodiments, innate immunogenicity is reduced by a 100-fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor.

The term “exhibits significantly less innate immunogenicity” refers to a detectable decrease in innate immunogenicity. In some embodiments, the term refers to a decrease such that an effective amount of the non-immunogenic RNA can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a decrease such that the non-immunogenic RNA can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In some embodiments, the decrease is such that the non-immunogenic RNA can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA. “Immunogenicity” is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal. The innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system.

As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence.

As used herein, the terms “linked,” “fused”, or “fusion” are used interchangeably. These terms refer to the joining together of two or more elements or components or domains.

Codon Optimization

In some embodiments, a payload (e.g., a polypeptide) described herein is encoded by a coding sequence which is codon-optimized and/or the G/C content of which is increased compared to wild type coding sequence. In some embodiments, one or more sequence regions of the coding sequence are codon-optimized and/or increased in the G/C content compared to the corresponding sequence regions of the wild type coding sequence. In some embodiments, codon-optimization and/or increased the G/C content does not change the sequence of the encoded amino acid sequence.

The term “codon-optimized” is understood by those in the art to refer to alteration of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, coding regions are preferably codon-optimized for optimal expression in a subject to be treated using an RNA polynucleotide described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of “rare codons”.

In some embodiments, guanosine/cytidine (G/C) content of a coding region (e.g., of a payload sequence) of an RNA is increased compared to the G/C content of the corresponding coding sequence of a wild type RNA encoding the payload, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that mRNA. Sequences having an increased G (guanosine)/C (cytidine) content are more stable than sequences having an increased A (adenosine)/U (uridine) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favourable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by the RNA, there are various possibilities for modification of the RNA sequence, compared to its wild type sequence. In particular, codons which contain A and/or U nucleosides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleosides.

In some embodiments, G/C content of a coding region of an RNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G/C content of a coding region of a wild type RNA.

5′ Cap

RNA capping is well researched and is described, e.g., in Decroly E et al. (2012) Nature Reviews 10: 51-65; and in Ramanathan A. et al., (2016) Nucleic Acids Res; 44(16): 7511-7526, the entire contents of each of which is hereby incorporated by reference. 5′ caps include a Cap-0 (also referred herein as “Cap0”), a Cap-1 (also referred herein as “Cap1”), or Cap-2 (also referred herein as “Cap2”). See, e.g., FIG. 1 of Ramanathan A et al., and FIG. 1 of Decroly E et al.

The term “5′-cap” as used herein refers to a structure found on the 5′-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5′- to 5′-triphosphate linkage (also referred to as Gppp or G(5′)ppp(5′)). In some embodiments, a guanosine nucleoside included in a 5′ cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on a base (guanine), and/or by methylation at one or more positions of a ribose. In some embodiments, a guanosine nucleoside included in a 5′ cap comprises a 3′O methylation at a ribose (3′OMeG). In some embodiments, a guanosine nucleoside included in a 5′ cap comprises methylation at the 7-position of guanine (m7G). In some embodiments, a guanosine nucleoside included in a 5′ cap comprises methylation at the 7-position of guanine and a 3′ O methylation at a ribose (m7(3′OMeG)).

In some embodiments, providing an RNA with a 5′-cap disclosed herein or a 5′-cap analog may be achieved by in vitro transcription, in which a 5′-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes. In some embodiments, co-transcriptional capping with a cap disclosed herein, e.g., with a cap1 or a cap1 analog, improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator. In some embodiments, improving capping efficiency can increase a translation efficiency and/or translation rate of an RNA, and/or increase expression of an encoded polypeptide.

In some embodiments, an RNA described herein comprises a 5′-cap or a 5′ cap analog, e.g., a Cap0, a Cap1 or a Cap2. In some embodiments, a provided RNA does not have uncapped 5′-triphosphates. In some embodiments, an RNA may be capped with a 5′-cap analog. In some embodiments, an RNA described herein comprises a Cap0. In some embodiments, an RNA described herein comprises a Cap1, e.g., as described herein. In some embodiments, an RNA described herein comprises a Cap2.

In some embodiments, a Cap0 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G). In some embodiments, a Cap0 structure is connected to an RNA via a 5′- to 5′-triphosphate linkage and is also referred to herein as m7Gppp or m7G(5′)ppp(5′).

In some embodiments, a Cap1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G) and a 2′O methylated first nucleotide in an RNA (2′OMeN₁). In some embodiments, a Cap1 structure is connected to an RNA via a 5′- to 5′-triphosphate linkage and is also referred to herein as m7Gppp(2′OMeN₁) or m7G(5′)ppp(5′)(2′OMeN₁). In some embodiments, N₁ is chosen from A, C, G, or U. In some embodiments, N₁ is A. In some embodiments, N₁ is C. In some embodiments, N₁ is G. In some embodiments, N₁ is U.

In some embodiments, a m7G(5′)ppp(5′)(2′OMeN₁) Cap1 structure comprises a second nucleotide, N₂ which is a cap proximal nucleotide at position 2 and is chosen from A, G, C, or U (m7G(5′)ppp(5′)(2′OMeN₁)N₂). In some embodiments, N₂ is A. In some embodiments, N₂ is C. In some embodiments, N₂ is G. In some embodiments, N₂ is U.

In some embodiments, a cap1 structure is or comprises m7G(5′)ppp(5′)(2′OMeA₁)pG₂ wherein A is a cap proximal nucleotide at position +1 and G is a cap proximal nucleotide at position +2, and has the following structure:

In some embodiments, a cap1 structure is or comprises m7G(5′)ppp(5′)(2′OMeA₁)pU2 wherein A is a cap proximal nucleotide at position 1 and U is a cap proximal nucleotide at position 2, and has the following structure:

In some embodiments, a cap1 structure is or comprises m7G(5′)ppp(5′)(2′OMeG₁)pG₂ wherein G is a cap proximal nucleotide at position 1 and G is a cap proximal nucleotide at position 2, and has the following structure:

In some embodiments, a Cap1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G) and one or more additional modifications, e.g., methylation on a ribose, and a 2′O methylated first nucleotide in an RNA. In some embodiments, a Cap1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine and a 3′O methylation at a ribose (m7(3′OMeG)); and a 2′O methylated first nucleotide in an RNA (2′OMeN₁). In some embodiments, a Cap1 structure is connected to an RNA via a 5′- to 5′-triphosphate linkage and is also referred to herein as m7(3′OMeG)ppp(2′OMeN₁) or m7(3′OMeG)(5′)ppp(5′)(2′OMeN₁). In some embodiments, N₁ is chosen from A, C, G, or U. In some embodiments, N₁ is A. In some embodiments, N₁ is C. In some embodiments, N₁ is G. In some embodiments, N₁ is U.

In some embodiments, a m7(3′oMeG)(5′)ppp(5′)(2′OMeN₁) Cap1 structure comprises a second nucleotide, N₂ which is a cap proximal nucleotide at position 2 and is chosen from A, G, C, or U (m7(3′OMeG)(5′)ppp(5′)(2′OMeN₁)N₂). In some embodiments, N₂ is A. In some embodiments, N₂ is C. In some embodiments, N₂ is G. In some embodiments, N₂ is U.

In some embodiments, a cap1 structure is or comprises m7(3′OMeG)(5′)ppp(5′)(2′OMeA₁)pG₂ wherein A is a cap proximal nucleotide at position 1 and G is a cap proximal nucleotide at position 2, and has the following structure:

In some embodiments, a cap1 structure is or comprises m7(3′OMeG)(5′)ppp(5′)(2′OMeG₁)pG₂ wherein G is a cap proximal nucleotide at position 1 and G is a cap proximal nucleotide at position 2, and has the following structure:

In some embodiments, a second nucleotide in a Cap1 structure can comprise one or more modifications, e.g., methylation. In some embodiments, a Cap1 structure comprising a second nucleotide comprising a 2′O methylation is a Cap2 structure.

In some embodiments, an RNA polynucleotide comprising a Cap1 structure has increased translation efficiency, increased translation rate and/or increased expression of an encoded payload relative to an appropriate reference comparator. In some embodiments, an RNA polynucleotide comprising a cap1 structure having m7(3′OMeG)(5′)ppp(5′)(2′OMeA₁)pG₂ wherein A is a cap proximal nucleotide at position 1 and G is a cap proximal nucleotide at position 2, has increased translation efficiency relative to an RNA polynucleotide comprising a cap1 structure having m7(3′OMeG)(5′)ppp(5′)(2′OMeG₁)pG₂ wherein G₁ is a cap proximal nucleotide at position 1 and G₂ is a cap proximal nucleotide at position 2. In some embodiments, increased translation efficiency is assessed upon administration of an RNA polynucleotide to a cell or an organism.

In some embodiments, a cap analog used in an RNA polynucleotide is m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG (also sometimes referred to as m₂ ^(7,3′O)G(5′)ppp(5′)m^(2′-O) ApG or m7(3′OMeG)(5′)ppp(5′)(2′OMeA)pG), which has the following structure:

Below is an exemplary Cap1 RNA, which comprises RNA and m₂ ^(7,3′O)G(5′)ppp(5′)m^(2′-O)ApG:

Below is another exemplary Cap1 RNA:

In some embodiments, an RNA polynucleotide disclosed herein comprises a Cap shown in any one of FIGS. 3A-3I. In some embodiments, an RNA polynucleotide disclosed herein comprises a Cap shown in FIG. 3A. In some embodiments, an RNA polynucleotide disclosed herein comprises a Cap shown in FIG. 3B. In some embodiments, an RNA polynucleotide disclosed herein comprises a Cap shown in FIG. 3C. In some embodiments, an RNA polynucleotide disclosed herein comprises a Cap shown in FIG. 3D. In some embodiments, an RNA polynucleotide disclosed herein comprises a Cap shown in FIG. 3E. In some embodiments, an RNA polynucleotide disclosed herein comprises a Cap shown in FIG. 3F. In some embodiments, an RNA polynucleotide disclosed herein comprises a Cap shown in FIG. 3G. In some embodiments, an RNA polynucleotide disclosed herein comprises a Cap shown in FIG. 3H. In some embodiments, an RNA polynucleotide disclosed herein comprises a Cap shown in FIG. 3I.

5′ UTR and Cap Proximal Sequences

In some embodiments, an RNA disclosed herein comprises a 5′-UTR. The term “untranslated region” or “UTR” relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) can be present 5′ (upstream) of an open reading frame (5′-UTR) and/or 3′ (downstream) of an open reading frame (3′-UTR). A 5′-UTR, if present, is located at the 5′ end, upstream of the start codon of a protein-encoding region. A 5′-UTR is downstream of the 5′-cap (if present), e.g. directly adjacent to the 5′-cap.

In some embodiments, a 5′ UTR disclosed herein comprises a cap proximal sequence, e.g., as disclosed herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5′ cap. In some embodiments, a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.

In some embodiments, a Cap structure comprises one or more polynucleotides of a cap proximal sequence. In some embodiments, a Cap structure comprises an m7 Guanosine cap and nucleotide+1 (N₁) of an RNA polynucleotide. In some embodiments, a Cap structure comprises an m7 Guanosine cap and nucleotide+2 (N₂) of an RNA polynucleotide. In some embodiments, a Cap structure comprises an m7 Guanosine cap and nucleotides+1 and +2 (N₁ and N₂) of an RNA polynucleotide.

Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, one or more residues of a cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and/or +5) may be included in an RNA by virtue of having been included in a cap entity that (e.g., a Cap1 structure, etc); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase). For example, in certain exemplified embodiments where a m₂ ^(7,3′-O)Gppp(m₁ ^(2′-O))ApG cap is utilized, +1 and +2 are the (m₁ ^(2′-O))A and G residues of the cap, and +3, +4, and +5 are added by polymerase (e.g., T7 polymerase).

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, wherein N₁ and N₂ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A. In some embodiments, N₁ is C. In some embodiments, N₁ is G. In some embodiments, N₁ is U. In some embodiments, N₂ is A. In some embodiments, N₂ is C. In some embodiments, N₂ is G. In some embodiments, N₂ is U.

In some embodiments, N₁ is A and N₂ is A. In some embodiments, N₁ is A and N₂ is C. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₁ is A and N₂ is U.

In some embodiments, N₁ is C and N₂ is A. In some embodiments, N₁ is C and N₂ is C. In some embodiments, N₁ is C and N₂ is G. In some embodiments, N₁ is C and N₂ is U.

In some embodiments, N₁ is G and N₂ is A. In some embodiments, N₁ is G and N₂ is C. In some embodiments, N₁ is G and N₂ is G. In some embodiments, N₁ is G and N₂ is U.

In some embodiments, N₁ is U and N₂ is A. In some embodiments, N₁ is U and N₂ is C. In some embodiments, N₁ is U and N₂ is G. In some embodiments, N₁ is U and N₂ is U.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure and N₃, N₄ and N₅, wherein N₁ to N₅ correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is A. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is C. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is G. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is U. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is A. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is G. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is C. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is U. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is A. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is C. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is G. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is U. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is A. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is C. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is G. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is A. In some embodiments, N₄ is U. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is A. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is C. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is G. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is U. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is A. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is G. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is C. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is U. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is A. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is C. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is G. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is U. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is A. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is C. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is G. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is C. In some embodiments, N₄ is U. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is A. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is C. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is G. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is U. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is A. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is G. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is C. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is U. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is A. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is C. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is G. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is U. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is A. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is C. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is G. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is G. In some embodiments, N₄ is U. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is A. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is C. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is G. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is U. In some embodiments, N₅ is A.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is A. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is G. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is C. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is U. In some embodiments, N₅ is G.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is A. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is C. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is G. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is U. In some embodiments, N₅ is C.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is A. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is C. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is G. In some embodiments, N₅ is U.

In some embodiments, N₁, N₂, N₃, N₄, or N₅ are any nucleotide, e.g., A, C, G or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₃ is U. In some embodiments, N₄ is U. In some embodiments, N₅ is U.

In some embodiments, a 5′ UTR disclosed herein comprises a cap proximal sequence, e.g., as disclosed herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5′ cap. In some embodiments, a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.

In some embodiments, a Cap structure comprises one or more polynucleotides of a cap proximal sequence. In some embodiments, a Cap structure comprises an m7 Guanosine cap and nucleotide+1 (N₁) of an RNA polynucleotide. In some embodiments, a Cap structure comprises an m7 Guanosine cap and nucleotide+2 (N₂) of an RNA polynucleotide. In some embodiments, a Cap structure comprises an m7 Guanosine cap and nucleotides+1 and +2 (N₁ and N₂) of an RNA polynucleotide.

In some embodiments, N₁ and N₂ are each independently chosen from: A, C, G, or U. In some embodiments, N₁ is A. In some embodiments, N₁ is C. In some embodiments, N₁ is G. In some embodiments, N₁ is U. In some embodiments, N₂ is A. In some embodiments, N₂ is C. In some embodiments, N₂ is G. In some embodiments, N₂ is U.

In some embodiments, N₁ and N₂ are each independently chosen from: A, C, G, or U. In some embodiments, N₁ is A. In some embodiments, N₁ is C. In some embodiments, N₁ is G. In some embodiments, N₁ is U. In some embodiments, N₂ is A. In some embodiments, N₂ is C. In some embodiments, N₂ is G. In some embodiments, N₂ is U.

In some embodiments, N₁ is A and N₂ is A. In some embodiments, N₁ is A and N₂ is C. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₁ is A and N₂ is U.

In some embodiments, N₁ is C and N₂ is A. In some embodiments, N₁ is C and N₂ is C. In some embodiments, N₁ is C and N₂ is G. In some embodiments, N₁ is C and N₂ is U.

In some embodiments, N₁ is G and N₂ is A. In some embodiments, N₁ is G and N₂ is C. In some embodiments, N₁ is G and N₂ is G. In some embodiments, N₁ is G and N₂ is U.

In some embodiments, N₁ is U and N₂ is A. In some embodiments, N₁ is U and N₂ is C. In some embodiments, N₁ is U and N₂ is G. In some embodiments, N₁ is U and N₂ is U.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising: A₃A₄X₅ (SEQ ID NO: 1). In some embodiments, N₁ and N₂ are each independently chosen from: A, C, G, or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, X₅ is chosen from A, C, G or U. In some embodiments, X₅ is A. In some embodiments, X₅ is C. In some embodiments, X₅ is G. In some embodiments, X₅ is U.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising: C₃A₄X₅ (SEQ ID NO: 2). In some embodiments, N₁ and N₂ are each independently chosen from: A, C, G, or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, X₅ is chosen from A, C, G or U. In some embodiments, X₅ is A. In some embodiments, X₅ is C. In some embodiments, X₅ is G. In some embodiments, X₅ is U.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising X₃Y₄X₅ (SEQ ID NO: 7). In some embodiments, N₁ and N₂ are each independently chosen from: A, C, G, or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, X₃ and X₅ is each independently chosen from A, C, G or U. In some embodiments, X₃ and/or X₅ is A. In some embodiments, X₃ and/or X₅ is C. In some embodiments, X₃ and/or X₅ is G. In some embodiments, X₃ and/or X₅ is U. In some embodiments, Y₄ is not C. In some embodiments, Y₄ is A. In some embodiments, Y₄ is G. In some embodiments, Y₄ is U.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising X₃Y₄X₅ (SEQ ID NO: 7). In some embodiments, N₁ and N₂ are each independently chosen from: A, C, G, or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, X₃ and X₅ is each independently chosen from A, C, G or U. In some embodiments, X₃ and/or X₅ is A. In some embodiments, X₃ and/or X₅ is C. In some embodiments, X₃ and/or X₅ is G. In some embodiments, X₃ and/or X₅ is U. In some embodiments, Y₄ is not G. In some embodiments, Y₄ is A. In some embodiments, Y₄ is C. In some embodiments, Y₄ is U.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising A₃C₄A₅ (SEQ ID NO: 3). In some embodiments, N₁ and N₂ are each independently chosen from: A, C, G, or U. In some embodiments, N₁ is A and N₂ is G.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising A₃U₄G₅ (SEQ ID NO: 4). In some embodiments, N₁ and N₂ are each independently chosen from: A, C, G, or U. In some embodiments, N₁ is A and N₂ is G.

In some embodiments, a Cap structure comprises one or more polynucleotides of a cap proximal sequence. In some embodiments, a Cap structure comprises an m7 Guanosine cap and nucleotide+1 (N₁) of an RNA polynucleotide. In some embodiments, a Cap structure comprises an m7 Guanosine cap and nucleotide+2 (N₂) of an RNA polynucleotide. In some embodiments, a Cap structure comprises an m7 Guanosine cap and nucleotides+1 and +2 (N₁ and N₂) of an RNA polynucleotide.

In some embodiments, N₁ and N₂ are any nucleotide, e.g., A, C, G, or U. In some embodiments, N₁ is A. In some embodiments, N₁ is C. In some embodiments, N₁ is G. In some embodiments, N₁ is U. In some embodiments, N₂ is A. In some embodiments, N₂ is C. In some embodiments, N₂ is G. In some embodiments, N₂ is U.

In some embodiments, N₁ and N₂ are any nucleotide, e.g., A, C, G, or U. In some embodiments, N₁ is A. In some embodiments, N₁ is C. In some embodiments, N₁ is G. In some embodiments, N₁ is U. In some embodiments, N₂ is A. In some embodiments, N₂ is C. In some embodiments, N₂ is G. In some embodiments, N₂ is U.

In some embodiments, N₁ is A and N₂ is A. In some embodiments, N₁ is A and N₂ is C. In some embodiments, N₁ is A and N₂ is G. In some embodiments, N₁ is A and N₂ is U.

In some embodiments, N₁ is C and N₂ is A. In some embodiments, N₁ is C and N₂ is C. In some embodiments, N₁ is C and N₂ is G. In some embodiments, N₁ is C and N₂ is U.

In some embodiments, N₁ is G and N₂ is A. In some embodiments, N₁ is G and N₂ is C. In some embodiments, N₁ is G and N₂ is G. In some embodiments, N₁ is G and N₂ is U.

In some embodiments, N₁ is U and N₂ is A. In some embodiments, N₁ is U and N₂ is C. In some embodiments, N₁ is U and N₂ is G. In some embodiments, N₁ is U and N₂ is U.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising: A₃A₄X₅ (SEQ ID NO: 1). In some embodiments, N₁ and N₂ are any nucleotide, e.g., A, C, G, or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, X₅ is chosen from A, C, G or U. In some embodiments, X₅ is A. In some embodiments, X₅ is C. In some embodiments, X₅ is G. In some embodiments, X₅ is U.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising: C₃A₄X₅ (SEQ ID NO: 2). In some embodiments, N₁ and N₂ are any nucleotide, e.g., A, C, G, or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, X₅ is any nucleotide, e.g., A, C, G or U. In some embodiments, X₅ is A. In some embodiments, X₅ is C. In some embodiments, X₅ is G. In some embodiments, X₅ is U.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising X₃Y₄X₅ (SEQ ID NO: 7). In some embodiments, N₁ and N₂ are any nucleotide, e.g., A, C, G, or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, X₃ and X₅ is any nucleotide, e.g., A, C, G or U. In some embodiments, X₃ and/or X₅ is A. In some embodiments, X₃ and/or X₅ is C. In some embodiments, X₃ and/or X₅ is G. In some embodiments, X₃ and/or X₅ is U. In some embodiments, Y₄ is not C. In some embodiments, Y₄ is A. In some embodiments, Y₄ is G. In some embodiments, Y₄ is U.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising X₃Y₄X₅ (SEQ ID NO: 7). In some embodiments, N₁ and N₂ are any nucleotide, e.g., A, C, G, or U. In some embodiments, N₁ is A and N₂ is G. In some embodiments, X₃ and X₅ is any nucleotide, e.g., A, C, G or U. In some embodiments, X₃ and/or X₅ is A. In some embodiments, X₃ and/or X₅ is C. In some embodiments, X₃ and/or X₅ is G. In some embodiments, X₃ and/or X₅ is U. In some embodiments, Y₄ is not G. In some embodiments, Y₄ is A. In some embodiments, Y₄ is C. In some embodiments, Y₄ is U.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising A₃C₄A₅ (SEQ ID NO: 3). In some embodiments, N₁ and N₂ are any nucleotide, e.g., A, C, G, or U. In some embodiments, N₁ is A and N₂ is G.

In some embodiments, a cap proximal sequence comprises N₁ and N₂ of a Cap structure, and a sequence comprising A₃U₄G₅ (SEQ ID NO: 4). In some embodiments, N₁ and N₂ are any nucleotide, e.g., A, C, G, or U. In some embodiments, N₁ is A and N₂ is G.

Exemplary 5′ UTRs include a human alpha globin (hAg) 5′UTR or a fragment thereof, a TEV 5′ UTR or a fragment thereof, a HSP70 5′ UTR or a fragment thereof, or a c-Jun 5′ UTR or a fragment thereof.

In some embodiments, an RNA disclosed herein comprises a hAg 5′ UTR or a fragment thereof. In some embodiments, an RNA disclosed herein comprises a hAg 5′ UTR having 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a human alpha globin 5′ UTR provided in SEQ ID NO: 11. In some embodiments, an RNA disclosed herein comprises a hAg 5′ UTR provided in SEQ ID NO: 11. In some embodiments, an RNA disclosed herein comprises a hAg 5′ UTR having 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a human alpha globin 5′ UTR provided in SEQ ID NO: 12. In some embodiments, an RNA disclosed herein comprises a hAg 5′ UTR provided in SEQ ID NO: 12.

3′ UTR

In some embodiments, an RNA disclosed herein comprises a 3′-UTR. A 3′-UTR, if present, is located at the 3′ end, downstream of the termination codon of a protein-encoding region, but the term “3′-UTR” does preferably not include the poly(A) sequence. Thus, the 3′-UTR is upstream of the poly(A) sequence (if present), e.g. directly adjacent to the poly(A) sequence.

In some embodiments, an RNA disclosed herein comprises a 3′ UTR comprising an F element and/or an I element. In some embodiments, a 3′ UTR or a proximal sequence thereto comprises a restriction site. In some embodiments, a restriction site is a BamHI site. In some embodiments, a restriction site is a XhoI site.

In some embodiments, an RNA disclosed herein comprises a 3′ UTR having 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a 3′ UTR provided in SEQ ID NO: 13. In some embodiments, an RNA disclosed herein comprises a 3′ UTR provided in SEQ ID NO: 13.

PolyA

In some embodiments, an RNA disclosed herein comprises a polyadenylate (PolyA) sequence, e.g., as described herein. In some embodiments, a PolyA sequence is situated downstream of a 3′-UTR, e.g., adjacent to a 3′-UTR.

As used herein, the term “poly(A) sequence” or “poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3′-end of an RNA polynucleotide. Poly(A) sequences are known to those of skill in the art and may follow the 3′-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. RNAs disclosed herein can have a poly(A) sequence attached to the free 3′-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5′) of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

The poly(A) sequence may be of any length. In some embodiments, a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, “essentially consists of” means that most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, “consists of” means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides. The term “A nucleotide” or “A” refers to adenylate.

In some embodiments, a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is referred to as poly(A) cassette.

In some embodiments, the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016/005324 A1, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 A1 may be used in the present invention. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. In some embodiments, the poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.

In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its 3′-end, i.e., the poly(A) sequence is not masked or followed at its 3′-end by a nucleotide other than A.

In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

In some embodiments, an RNA disclosed herein comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO: 14, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, an RNA disclosed herein comprises a poly(A) sequence of SEQ ID NO: 14.

Payloads

In some embodiments, an RNA polynucleotide disclosed herein comprises a sequence encoding a payload, e.g., as described herein. In some embodiments, a sequence encoding a payload comprises a promoter sequence. In some embodiments, a sequence encoding a payload comprises a sequence encoding a secretory signal peptide.

In some embodiments, a payload is chosen from: a protein replacement polypeptide; an antibody agent; a cytokine; an antigenic polypeptide; a gene editing component; a regenerative medicine component or combinations thereof.

In some embodiments, a payload is or comprises a protein replacement polypeptide. In some embodiments, a protein replacement polypeptide comprises a polypeptide with aberrant expression in a disease or disorder. In some embodiments, a protein replacement polypeptide comprises an intracellular protein, an extracellular protein, or a transmembrane protein. In some embodiments, a protein replacement polypeptide comprises an enzyme.

In some embodiments, a disease or disorder with aberrant expression of a polypeptide includes but is not limited to: a rare disease, a metabolic disorder, a muscular dystrophy, a cardiovascular disease, or a monogenic disease.

In some embodiments, a payload is or comprises an antibody agent. In some embodiments, an antibody agent binds to a polypeptide expressed on a cell. In some embodiments, an antibody agent comprises a CD3 antibody, a Claudin 6 antibody, or a combination thereof.

In some embodiments, a payload is or comprises a cytokine or a fragment or a variant thereof. In some embodiments, a cytokine comprises: IL-12 or a fragment or variant or a fusion thereof, IL-15 or a fragment or a variant or a fusion thereof, GMCSF or a fragment or a variant thereof; or IFN-alpha or a fragment or a variant thereof.

In some embodiments, a payload is or comprises an antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof. In some embodiments, an antigenic polypeptide comprises one epitope from an antigen. In some embodiments, an antigenic polypeptide comprises a plurality of distinct epitopes from an antigen. In some embodiments, an antigenic polypeptide comprising a plurality of distinct epitopes from an antigen is polyepitopic.

In some embodiments, an antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or a self-antigenic polypeptide.

In some embodiments, a viral antigenic polypeptide comprises an HIV antigenic polypeptide, an influenza antigenic polypeptide, a Coronavirus antigenic polypeptide, a Rabies antigenic polypeptide, or a Zika virus antigenic polypeptide.

In some embodiments, a viral antigenic polypeptide is or comprises a Coronavirus antigenic polypeptide. In some embodiments, a Coronavirus antigen is or comprises a SARS-CoV-2 protein. In some embodiments, a SARS-CoV-2 protein comprises a SARS-CoV-2 Spike (S) protein, or an immunogenic variant or an immunogenic fragment thereof. In some embodiments, a SARS-CoV-2 protein, or immunogenic variant or immunogenic fragment thereof, comprises proline residues at positions 986 and 987.

In some embodiments, a SARS-CoV-2 S polypeptide has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a SARS-CoV-2 S polypeptide disclosed herein. In some embodiments, a SARS-CoV-2 S polypeptide has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 9.

In some embodiments, a SARS-CoV-2 S polypeptide is encoded by an RNA having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to a SARS-CoV-2 S polynucleotide disclosed herein. In some embodiments, a SARS-CoV-2 S polypeptide is encoded by an RNA having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 10.

In some embodiments, a payload is or comprises a tumor antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof. In some embodiments, a tumor antigenic polypeptide comprises a tumor specific antigen, a tumor associated antigen, a tumor neoantigen, or a combination thereof. In some embodiments, a tumor antigenic polypeptide comprises p53, ART-4, BAGE, ss-catenin/m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan-A, MC1R, Myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Plac-1, Pm1/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE, WT, WT-1, or a combination thereof.

In some embodiments, a tumor antigenic polypeptide comprises a tumor antigen from a carcinoma, a sarcoma, a melanoma, a lymphoma, a leukemia, or a combination thereof.

In some embodiments, a tumor antigenic polypeptide comprises a melanoma tumor antigen.

In some embodiments, a tumor antigenic polypeptide comprises a prostate cancer antigen.

In some embodiments, a tumor antigenic polypeptide comprises a HPV16 positive head and neck cancer antigen.

In some embodiments, a tumor antigenic polypeptide comprises a breast cancer antigen.

In some embodiments, a tumor antigenic polypeptide comprises an ovarian cancer antigen.

In some embodiments, a tumor antigenic polypeptide comprises a lung cancer antigen.

In some embodiments, a tumor antigenic polypeptide comprises an NSCLC antigen.

In some embodiments, a payload is or comprises a self-antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof. In some embodiments, a self-antigenic polypeptide comprises an antigen that is typically expressed on cells and is recognized as a self-antigen by an immune system. In some embodiments, a self-antigenic polypeptide comprises: a multiple sclerosis antigenic polypeptide, a Rheumatoid arthritis antigenic polypeptide, a lupus antigenic polypeptide, a celiac disease antigenic polypeptide, a Sjogren's syndrome antigenic polypeptide, or an ankylosing spondylitis antigenic polypeptide, or a combination thereof.

Exemplary Polynucleotides

In some embodiments, an RNA polynucleotide described herein or a composition or medical preparation comprising the same comprises a nucleotide sequence disclosed herein. In some embodiments, an RNA polynucleotide comprises a sequence having at least 80% identity to a nucleotide sequence disclosed herein. In some embodiments, an RNA polynucleotide comprises a sequence encoding a polypeptide having at least 80% identity to a polypeptide sequence disclosed herein. Exemplary nucleotide and polypeptide sequences are provided e.g., in Table 1 or in this section titled “Exemplary polynucleotides” or in Example 2.

In some embodiments, an RNA polynucleotide described herein or a composition or medical preparation comprising the same is transcribed by a DNA template. In some embodiments, a DNA template used to transcribe an RNA polynucleotide described herein comprises a sequence complementary to an RNA polynucleotide.

In some embodiments, a payload described herein is encoded by an RNA polynucleotide described herein comprising a nucleotide sequence disclosed herein, e.g., in Table 1 or in this section titled “Exemplary polynucleotides” or in Example 2. In some embodiments, an RNA polynucleotide encodes a polypeptide payload having at least 80% identity to a polypeptide payload sequence disclosed herein. In some embodiments, a payload described herein is encoded by an RNA polynucleotide transcribed by a DNA template comprising a sequence complementary to an RNA polynucleotide.

TABLE 1 Exemplary sequences of RNA constructs disclosed herein SEQ ID Sequence NO information Sequence 1 Cap proximal a₃a₄x₅ consensus wherein X₅ is A, G, C, or U sequence (nucleotides 1-5 of a 5′ UTR) 2 Cap proximal C₃A₄X₅ consensus wherein X₅ is A, G, C, or U sequence (nucleotides +3 to +5 of a 5′ UTR) 3 Cap proximal A₃C₄A₅ sequence (nucleotides +3 to +5 of a 5′ UTR) 4 Cap proximal A₃U₄G₅ sequence (nucleotides +3 to +5 of a 5′ UTR) 5 Cap proximal A₃A₄U₅ sequence (nucleotides +3 to +5 of a 5′ UTR) 6 Cap proximal C₃A₄C₅ sequence (nucleotides +3 to +5 of a 5′ UTR) 7 Cap proximal X₃Y₄X₅ sequence wherein X₃ or X₅ are each (nucleotides independently chosen from +3 to A, G, C, or U; and +5 of a Y₄ is not C. 5′ UTR) 8 Ligation 3 GAGUCGCUAGCCGCGUCGCU sequence 9 S protein PP MFVFLVLLPLVSSQCVNLTTRTQLPP (amino acid) AYTNSFTRGVYYPDKVFRSSVLHST (V08/V09) QDLFLPFFSNVTWFHAIHVSGTNGT KRFDNPVLPFNDGVYFASTEKSNII RGWIFGTTLDSKTQSLLIVNNATNV VIKVCEFQFCNDPFLGVYYHKNNKS WMESEFRVYSSANNCTFEYVSQPFL MDLEGKQGNFKNLREFVFKNIDGYF KIYSKHTPINLVRDLPQGFSALEPL VDLPIGINITRFQTLLALHRSYLTP GDSSSGWTAGAAAYYVGYLQPRTFL LKYNENGTITDAVDCALDPLSETKC TLKSFTVEKGIYQTSNFRVQPTESI VRFPNITNLCPFGEVFNATRFASVY AWNRKRISNCVADYSVLYNSASFST FKCYGVSPTKLNDLCFTNVYADSFV IRGDEVRQIAPGQTGKIADYNYKLP DDFTGCVIAWNSNNLDSKVGGNYNY LYRLFRKSNLKPFERDISTEIYQAG STPCNGVEGFNCYFPLQSYGFQPTN GVGYQPYRVVVLSFELLHAPATVCG PKKSTNLVKNKCVNFNFNGLTGTGV LTESNKKFLPFQQFGRDIADTTDAV RDPQTLEILDITPCSFGGVSVITPG TNTSNQVAVLYQDVNCTEVPVAIHA DQLTPTWRVYSTGSNVFQTRAGCLI GAEHVNNSYECDIPIGAGICASYQT QTNSPRRARSVASQSIIAYTMSLGA ENSVAYSNNSIAIPTNFTISVTTEI LPVSMTKTSVDCTMYICGDSTECSN LLLQYGSFCTQLNRALTGIAVEQDK NTQEVFAQVKQIYKTPPIKDFGGFN FSQILPDPSKPSKRSFIEDLLFNKV TLADAGFIKQYGDCLGDIAARDLIC AQKFNGLTVLPPLLTDEMIAQYTSA LLAGTITSGWTFGAGAALQIPFAMQ MAYRFNGIGVTQNVLYENQKLIANQ FNSAIGKIQDSLSSTASALGKLQDV VNQNAQALNTLVKQLSSNFGAISSV LNDILSRLDPPEAEVQIDRLITGRL QSLQTYVTQQLIRAAEIRASANLAA TKMSECVLGQSKRVDFCGKGYHLMS FPQSAPHGVVFLHVTYVPAQEKNFT TAPAICFfDGKAHFPREGVFVSNGT FrWFVTQRNFYEPQIITTDNTFVSG NCDVVIGIVNNTVYDPLQPELDSFK EELDKYFKNHTSPDVDLGDISGINA SVVNIQKEIDRLNEVAKNLNESLID LQELGKYEQYIKWPWYIWLGFIAGL IAIVMVTIMLCCMTSCCSCLKGCCS CGSCCKFDEDDSEPVLKGVKLHYT 10 RBP020.2 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguucg uguuccuggu gcugcugccu cuggugucca gccagugugu gaaccugacc accagaacac agcugccucc agccuacacc aacagcuuua ccagaggcgu guacuacccc gacaaggugu ucagauccag cgugcugcac ucuacccagg accuguuccu gccuuucuuc agcaacguga ccugguucca cgccauccac guguccggca ccaauggcac caagagauuc gacaaccccg ugcugcccuu caacgacggg guguacuuug ccagcaccga gaaguccaac aucaucagag gcuggaucuu cggcaccaca cuggacagca agacccagag ccugcugauc gugaacaacg ccaccaacgu ggucaucaaa gugugcgagu uccaguucug caacgacccc uuccugggcg ucuacuacca caagaacaac aagagcugga uggaaagcga guuccgggug uacagcagcg ccaacaacug caccuucgag uacguguccc agccuuuccu gauggaccug gaaggcaagc agggcaacuu caagaaccug cgcgaguucg uguuuaagaa caucgacggc uacuucaaga ucuacagcaa gcacaccccu aucaaccucg ugcgggaucu gccucagggc uucucugcuc uggaaccccu gguggaucug cccaucggca ucaacaucac ccgguuucag acacugcugg cccugcacag aagcuaccug acaccuggcg auagcagcag cggauggaca gcuggugccg ccgcuuacua ugugggcuac cugcagccua gaaccuuccu gcugaaguac aacgagaacg gcaccaucac cgacgccgug gauugugcuc uggauccucu gagcgagaca aagugcaccc ugaaguccuu caccguggaa aagggcaucu accagaccag caacuuccgg gugcagccca ccgaauccau cgugcgguuc cccaauauca ccaaucugug ccccuucggc gagguguuca augccaccag auucgccucu guguacgccu ggaaccggaa gcggaucagc aauugcgugg ccgacuacuc cgugcuguac aacuccgcca gcuucagcac cuucaagugc uacggcgugu ccccuaccaa gcugaacgac cugugcuuca caaacgugua cgccgacagc uucgugaucc ggggagauga agugcggcag auugccccug gacagacagg caagaucgcc gacuacaacu acaagcugcc cgacgacuuc accggcugug ugauugccug gaacagcaac aaccuggacu ccaaagucgg cggcaacuac aauuaccugu accggcuguu ccggaagucc aaucugaagc ccuucgagcg ggacaucucc accgagaucu aucaggccgg cagcaccccu uguaacggcg uggaaggcuu caacugcuac uucccacugc aguccuacgg cuuucagccc acaaauggcg ugggcuauca gcccuacaga gugguggugc ugagcuucga acugcugcau gccccugcca cagugugcgg cccuaagaaa agcaccaauc ucgugaagaa caaaugcgug aacuucaacu ucaacggccu gaccggcacc ggcgugcuga cagagagcaa caagaaguuc cugccauucc agcaguuugg ccgggauauc gccgauacca cagacgccgu uagagauccc cagacacugg aaauccugga caucaccccu ugcagcuucg gcggaguguc ugugaucacc ccuggcacca acaccagcaa ucagguggca gugcuguacc aggacgugaa cuguaccgaa gugcccgugg ccauucacgc cgaucagcug acaccuacau ggcgggugua cuccaccggc agcaaugugu uucagaccag agccggcugu cugaucggag ccgagcacgu gaacaauagc uacgagugcg acauccccau cggcgcugga aucugcgcca gcuaccagac acagacaaac agcccucgga gagccagaag cguggccagc cagagcauca uugccuacac aaugucucug ggcgccgaga acagcguggc cuacuccaac aacucuaucg cuauccccac caacuucacc aucagcguga ccacagagau ccugccugug uccaugacca agaccagcgu ggacugcacc auguacaucu gcggcgauuc caccgagugc uccaaccugc ugcugcagua cggcagcuuc ugcacccagc ugaauagagc ccugacaggg aucgccgugg aacaggacaa gaacacccaa gagguguucg cccaagugaa gcagaucuac aagaccccuc cuaucaagga cuucggcggc uucaauuuca gccagauucu gcccgauccu agcaagccca gcaagcggag cuucaucgag gaccugcugu ucaacaaagu gacacuggcc gacgccggcu ucaucaagca guauggcgau ugucugggcg acauugccgc cagggaucug auuugcgccc agaaguuuaa cggacugaca gugcugccuc cucugcugac cgaugagaug aucgcccagu acacaucugc ccugcuggcc ggcacaauca caagcggcug gacauuugga gcaggcgccg cucugcagau ccccuuugcu augcagaugg ccuaccgguu caacggcauc ggagugaccc agaaugugcu guacgagaac cagaagcuga ucgccaacca guucaacagc gccaucggca agauccagga cagccugagc agcacagcaa gcgcccuggg aaagcugcag gacgugguca accagaaugc ccaggcacug aacacccugg ucaagcagcu guccuccaac uucggcgcca ucagcucugu gcugaacgau auccugagca gacuggaccc uccugaggcc gaggugcaga ucgacagacu gaucacaggc agacugcaga gccuccagac auacgugacc cagcagcuga ucagagccgc cgagauuaga gccucugcca aucuggccgc caccaagaug ucugagugug ugcugggcca gagcaagaga guggacuuuu gcggcaaggg cuaccaccug augagcuucc cucagucugc cccucacggc gugguguuuc ugcacgugac auaugugccc gcucaagaga agaauuucac caccgcucca gccaucugcc acgacggcaa agcccacuuu ccuagagaag gcguguucgu guccaacggc acccauuggu ucgugacaca gcggaacuuc uacgagcccc agaucaucac caccgacaac accuucgugu cuggcaacug cgacgucgug aucggcauug ugaacaauac cguguacgac ccucugcagc ccgagcugga cagcuucaaa gaggaacugg acaaguacuu uaagaaccac acaagccccg acguggaccu gggcgauauc agcggaauca augccagcgu cgugaacauc cagaaagaga ucgaccggcu gaacgaggug gccaagaauc ugaacgagag ccugaucgac cugcaagaac uggggaagua cgagcaguac aucaaguggc ccugguacau cuggcugggc uuuaucgccg gacugauugc caucgugaug gucacaauca ugcuguguug caugaccagc ugcuguagcu gccugaaggg cuguuguagc uguggcagcu gcugcaaguu cgacgaggac gauucugagc ccgugcugaa gggcgugaaa cugcacuaca caugaugacu cgagcuggua cugcaugcac gcaaugcuag cugccccuuu cccguccugg guaccccgag ucucccccga ccucgggucc cagguaugcu cccaccucca ccugccccac ucaccaccuc ugcuaguucc agacaccucc caagcacgca gcaaugcagc ucaaaacgcu uagccuagcc acacccccac gggaaacagc agugauuaac cuuuagcaau aaacgaaagu uuaacuaagc uauacuaacc ccaggguugg ucaauuucgu gccagccaca cccuggagcu agcaaaaaaa aaaaaaaaaa aaaaaaaaaa aaagcauaug acuaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 11 Human alpha

AAACUAGUAUUCUUCUGGUCC globin CCACAGACUCAGAGAGAACCC 5′ UTR with (

first 5 nucleo- tides) 12 Human alpha AAACUAGUAUUCUUCUGGUCCCCA globin CAGACUCAGAGAGAACCC 5′ UTR (without first 5 nucleo- tides) 13 3′ UTR CUGGUACUGCAUGCACGCAAUGCUA GCUGCCCCUUUCCCGUCCUGGGUAC CCCGAGUCUCCCCCGACCUCGGGUC CCAGGUAUGCUCCCACCUCCACCUG CCCCACUCACCACCUCUGCUAGUUC CAGACACCUCCCAAGCACGCAGCAA UGCAGCUCAAAACGCUUAGCCUAGC CACACCCCCACGGGAAACAGCAGUG AUUAACCUUUAGCAAUAAACGAAAG UUUAACUAAGCUAUACUAACCCCAG GGUUGGUCAAUUUCGUGCCAGCCAC ACC 14 A30L70 AAAAAAAAAAAAAAAAAAAAAAAAA PolyA AAAAAGCAUAUGACUAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAA 15 Exemplary

AAACUAGUAUUCUUCUGGU RNA CCCCACAGACUCAGAGAGAACCC GCC poly- ACCCUCGAG CUGGUACUGCAUGCAC nucleotide GCAAUGCUAGCUGCCCCUUUCCCGU (vA3.0.1) CCUGGGUACCCCGAGUCUCCCCCGA without CCUCGGGUCCCAGGUAUGCUCCCAC coding CUCCACCUGCCCCACUCACCACCUC sequence of UGCUAGUUCCAGACACCUCCCAAGC a payload ACGCAGCAAUGCAGCUCAAAACGCU Bold = UAGCCUAGCCACACCCCCACGGGAA 5′ UTR ACAGCAGUGAUUAACCUUUAGCAAU Bold and AAACGAAAGUUUAACUAAGCUAUAC underlined = UAACCCCAGGGUUGGUCAAUUUCGU cap GCCAGCCACAC CCUGGAGCUAGC proximal AAAAAAAAAAAAAA sequence AAAAAAAAAAAAAAAAGCAUAUGAC Underlined = UAAAAAAAAAAAAAAAAAAAAAAAA Kozak AAAAAAAAAAAAAAAAAAAAAAAAA sequence AAAAAAAAAAAAAAAAAAAAA Italicized and underlined = 3′ UTR Italicized = PolyA 16 Exemplary- AGAAUAAACUAGUAUUCUUCUGGUC RNA CCCACAGACUCAGAGAGAACCCGCC poly- ACCAUGUUCGUGUUCCUGGUGCUGC nucleotide UGCCUCUGGUGUCCAGCCAGUGUGU (vA3.0.1) GAACCUGACCACCAGAACACAGCUG with a CCUCCAGCCUACACCAACAGCUUUA payload CCAGAGGCGUGUACUACCCCGACAA sequence GGUGUUCAGAUCCAGCGUGCUGCAC Underline = UCUACCCAGGACCUGUUCCUGCCUU exemplary UCUUCAGCAACGUGACCUGGUUCCA payload CGCCAUCCACGUGUCCGGCACCAAU sequence GGCACCAAGAGAUUCGACAACCCCG (BNT162b2) UGCUGCCCUUCAACGACGGGGUGUA CUUUGCCAGCACCGAGAAGUCCAAC AUCAUCAGAGGCUGGAUCUUCGGCA CCACACUGGACAGCAAGACCCAGAG CCUGCUGAUCGUGAACAACGCCACC AACGUGGUCAUCAAAGUGUGCGAGU UCCAGUUCUGCAACGACCCCUUCCU GGGCGUCUACUACCACAAGAACAAC AAGAGCUGGAUGGAAAGCGAGUUCC GGGUGUACAGCAGCGCCAACAACuG CACCUUCGAGUACGUGUCCCAGCCU UUCCUGAUGGACCUGGAAGGCAAGC AGGGCAACUUCAAGAACCUGCGCGA GUUCGUGUUUAAGAACAUCGACGGC UACUUCAAGAUCUACAGCAAGCACA CCCCUAUCAACCUCGUGCGGGAUCU GCCUCAGGGCUUCUCUGCUCUGGAA CCCCUGGUGGAUCUGCCCAUCGGCA UCAACAUCACCCGGUUUCAGACACU GCUGGCCCUGCACAGAAGCUACCUG ACACCUGGCGAUAGCAGCAGCGGAU GGACAGCUGGUGCCGCCGCUUACUA UGUGGGCUACCUGCAGCCUAGAACC UUCCUGCUGAAGUACAACGAGAACG GCACCAUCACCGACGCCGUGGAUUG UGCUCUGGAUCCUCUGAGCGAGACA AAGUGCACCCUGAAGUCCUUCACCG UGGAAAAGGGCAUCUACCAGACCAG CAACUUCCGGGUGCAGCCCACCGAA UCCAUCGUGCGGUUCCCCAAUAUCA CCAAUCUGUGCCCCUUCGGCGAGGU GUUCAAUGCCACCAGAUUCGCCUCU GUGUACGCCUGGAACCGGAAGCGGA UCAGCAAUUGCGUGGCCGACUACUC CGUGCUGUACAACUCCGCCAGCUUC AGCACCUUCAAGUGCUACGGCGUGU CCCCUACCAAGCUGAACGACCUGUG CUUCACAAACGUGUACGCCGACAGC UUCGUGAUCCGGGGAGAUGAAGUGC GGCAGAUUGCCCCUGGACAGACAGG CAAGAUCGCCGACUACAACUACAAG CUGCCCGACGACUUCACCGGCUGUG UGAUUGCCUGGAACAGCAACAACCU GGACUCCAAAGUCGGCGGCAACUAC AAUUACCUGUACCGGCUGUUCCGGA AGUCCAAUCUGAAGCCCUUCGAGCG GGACAUCUCCACCGAGAUCUAUCAG GCCGGCAGCACCCCUUGUAACGGCG UGGAAGGCUUCAACUGCUACUUCCC ACUGCAGUCCUACGGCUUUCAGCCC ACAAAUGGCGUGGGCUAUCAGCCCU ACAGAGUGGUGGUGCUGAGCUUCGA ACUGCUGCAUGCCCCUGCCACAGUG UGCGGCCCUAAGAAAAGCACCAAUC UCGUGAAGAACAAAUGCGUGAACUU CAACUUCAACGGCCUGACCGGCACC GGCGUGCUGACAGAGAGCAACAAGA AGUUCCUGCCAUUCCAGCAGUUUGG CCGGGAUAUCGCCGAUACCACAGAC GCCGUUAGAGAUCCCCAGACACUGG AAAUCCUGGACAUCACCCCUUGCAG CUUCGGCGGAGUGUCUGUGAUCACC CCUGGCACCAACACCAGCAAUCAGG UGGCAGUGCUGUACCAGGACGUGAA CUGUACCGAAGUGCCCGUGGCCAUU CACGCCGAUCAGCUGACACCUACAU GGCGGGUGUACUCCACCGGCAGCAA UGUGUUUCAGACCAGAGCCGGCUGU CUGAUCGGAGCCGAGCACGUGAACA AUAGCUACGAGUGCGACAUCCCCAU CGGCGCUGGAAUCUGCGCCAGCUAC CAGACACAGACAAACAGCCCUCGGA GAGCCAGAAGCGUGGCCAGCCAGAG CAUCAUUGCCUACACAAUGUCUCUG GGCGCCGAGAACAGCGUGGCCUACU CCAACAACUCUAUCGCUAUCCCCAC CAACUUCACCAUCAGCGUGACCACA GAGAUCCUGCCUGUGUCCAUGACCA AGACCAGCGUGGACUGCACCAUGUA CAUCUGCGGCGAUUCCACCGAGUGC UCCAACCUGCUGCUGCAGUACGGCA GCUUCUGCACCCAGCUGAAUAGAGC CCUGACAGGGAUCGCCGUGGAACAG GACAAGAACACCCAAGAGGUGUUCG CCCAAGUGAAGCAGAUCUACAAGAC CCCUCCUAUCAAGGACUUCGGCGGC UUCAAUUUCAGCCAGAUUCUGCCCG AUCCUAGCAAGCCCAGCAAGCGGAG CUUCAUCGAGGACCUGCUGUUCAAC AAAGUGACACUGGCCGACGCCGGCU UCAUCAAGCAGUAUGGCGAUUGUCU GGGCGACAUUGCCGCCAGGGAUCUG AUUUGCGCCCAGAAGUUUAACGGAC UGACAGUGCUGCCUCCUCUGCUGAC CGAUGAGAUGAUCGCCCAGUACACA UCUGCCCUGCUGGCCGGCACAAUCA CAAGCGGCUGGACAUUUGGAGCAGG CGCCGCUCUGCAGAUCCCCUUUGCU AUGCAGAUGGCCUACCGGUUCAACG GCAUCGGAGUGACCCAGAAUGUGCU GUACGAGAACCAGAAGCUGAUCGCC AACCAGUUCAACAGCGCCAUCGGCA AGAUCCAGGACAGCCUGAGCAGCAC AGCAAGCGCCCUGGGAAAGCUGCAG GACGUGGUCAACCAGAAUGCCCAGG CACUGAACACCCUGGUCAAGCAGCU GUCCUCCAACUUCGGCGCCAUCAGC UCUGUGCUGAACGAUAUCCUGAGCA GACUGGACCCUCCUGAGGCCGAGGU GCAGAUCGACAGACUGAUCACAGGC AGACUGCAGAGCCUCCAGACAUACG UGACCCAGCAGCUGAUCAGAGCCGC CGAGAUUAGAGCCUCUGCCAAUCUG GCCGCCACCAAGAUGUCUGAGUGUG UGCUGGGCCAGAGCAAGAGAGUGGA CUUUUGCGGCAAGGGCUACCACCUG AUGAGCUUCCCUCAGUCUGCCCCUC ACGGCGUGGUGUUUCUGCACGUGAC AUAUGUGCCCGCUCAAGAGAAGAAU UUCACCACCGCUCCAGCCAUCUGCC ACGACGGCAAAGCCCACUUUCCUAG AGAAGGCGUGUUCGUGUCCAACGGC ACCCAUUGGUUCGUGACACAGCGGA ACUUCUACGAGCCCCAGAUCAUCAC CACCGACAACACCUUCGUGUCUGGC AACUGCGACGUCGUGAUCGGCAUUG UGAACAAUACCGUGUACGACCCUCU GCAGCCCGAGCUGGACAGCUUCAAA GAGGAACUGGACAAGUACUUUAAGA ACCACACAAGCCCCGACGUGGACCU GGGCGAUAUCAGCGGAAUCAAUGCC AGCGUCGUGAACAUCCAGAAAGAGA UCGACCGGCUGAACGAGGUGGCCAA GAAUCUGAACGAGAGCCUGAUCGAC CUGCAAGAACUGGGGAAGUACGAGC AGUACAUCAAGUGGCCCUGGUACAU CUGGCUGGGCUUUAUCGCCGGACUG AUUGCCAUCGUGAUGGUCACAAUCA UGCUGUGUUGCAUGACCAGCUGCUG UAGCUGCCUGAAGGGCUGUUGUAGC UGUGGCAGCUGCUGCAAGUUCGACG AGGACGAUUCUGAGCCCGUGCUGAA GGGCGUGAAACUGCACUACACAUGA UGACUCGAGCUGGUACUGCAUGCAC GCAAUGCUAGCUGCCCCUUUCCCGU CCUGGGUACCCCGAGUCUCCCCCGA CCUCGGGUCCCAGGUAUGCUCCCAC CUCCACCUGCCCCACUCACCACCUC UGCUAGUUCCAGACACCUCCCAAGC ACGCAGCAAUGCAGCUCAAAACGCU UAGCCUAGCCACACCCCCACGGGAA ACAGCAGUGAUUAACCUUUAGCAAU AAACGAAAGUUUAACUAAGCUAUAC UAACCCCAGGGUUGGUCAAUUUCGU GCCAGCCACACCCUGGAGCUAGCAA AAAAAAAAAAAAAAAAAAAAAAAAA AAAGCAUAUGACUAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAA

RBL063.1 (SEQ ID NO: 28 nucleotide; SEQ ID NO: 9 amino acid) Structure  beta-S-ARCA(D1)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (S152 full-length protein, sequence variant) SEQ ID NO: 28 gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccauguuugu    60 guuucuugug cugcugccuc uugugucuuc ucagugugug aauuugacaa caagaacaca   120 gcugccacca gcuuauacaa auucuuuuac cagaggagug uauuauccug auaaaguguu   180 uagaucuucu gugcugcaca gcacacagga ccuguuucug ccauuuuuua gcaaugugac   240 augguuucau gcaauucaug ugucuggaac aaauggaaca aaaagauuug auaauccugu   300 gcugccuuuu aaugauggag uguauuuugc uucaacagaa aagucaaaua uuauuagagg   360 auggauuuuu ggaacaacac uggauucuaa aacacagucu cugcugauug ugaauaaugc   420 aacaaaugug gugauuaaag ugugugaauu ucaguuuugu aaugauccuu uucugggagu   480 guauuaucac aaaaauaaua aaucuuggau ggaaucugaa uuuagagugu auuccucugc   540 aaauaauugu acauuugaau augugucuca gccuuuucug auggaucugg aaggaaaaca   600 gggcaauuuu aaaaaucuga gagaauuugu guuuaaaaau auugauggau auuuuaaaau   660 uuauucuaaa cacacaccaa uuaauuuagu gagagaucug ccucagggau uuucugcucu   720 ggaaccucug guggaucugc caauuggcau uaauauuaca agauuucaga cacugcuggc   780 ucugcacaga ucuuaucuga caccuggaga uucuucuucu ggauggacag ccggagcugc   840 agcuuauuau gugggcuauc ugcagccaag aacauuucug cugaaauaua augaaaaugg   900 aacaauuaca gaugcugugg auugugcucu ggauccucug ucugaaacaa aauguacauu   960 aaaaucuuuu acaguggaaa aaggcauuua ucagacaucu aauuuuagag ugcagccaac  1020 agaaucuauu gugagauuuc caaauauuac aaaucugugu ccauuuggag aaguguuuaa  1080 ugcaacaaga uuugcaucug uguaugcaug gaauagaaaa agaauuucua auuguguggc  1140 ugauuauucu gugcuguaua auagugcuuc uuuuuccaca uuuaaauguu auggaguguc  1200 uccaacaaaa uuaaaugauu uauguuuuac aaauguguau gcugauucuu uugugaucag  1260 aggugaugaa gugagacaga uugcccccgg acagacagga aaaauugcug auuacaauua  1320 caaacugccu gaugauuuua caggaugugu gauugcuugg aauucuaaua auuuagauuc  1380 uaaaguggga ggaaauuaca auuaucugua cagacuguuu agaaaaucaa aucugaaacc  1440 uuuugaaaga gauauuucaa cagaaauuua ucaggcugga ucaacaccuu guaauggagu  1500 ggaaggauuu aauuguuauu uuccauuaca gagcuaugga uuucagccaa ccaauggugu  1560 gggauaucag ccauauagag ugguggugcu gucuuuugaa cugcugcaug caccugcaac  1620 agugugugga ccuaaaaaau cuacaaauuu agugaaaaau aaauguguga auuuuaauuu  1680 uaauggauua acaggaacag gagugcugac agaaucuaau aaaaaauuuc ugccuuuuca  1740 gcaguuuggc agagauauug cagauaccac agaugcagug agagauccuc agacauuaga  1800 aauucuggau auuacaccuu guucuuuugg gggugugucu gugauuacac cuggaacaaa  1860 uacaucuaau cagguggcug ugcuguauca ggaugugaau uguacagaag ugccaguggc  1920 aauucaugca gaucagcuga caccaacaug gagaguguau ucuacaggau cuaauguguu  1980 ucagacaaga gcaggauguc ugauuggagc agaacaugug aauaauucuu augaauguga  2040 uauuccaauu ggagcaggca uuugugcauc uuaucagaca cagacaaauu ccccaaggag  2100 agcaagaucu guggcaucuc agucuauuau ugcauacacc augucucugg gagcagaaaa  2160 uucuguggca uauucuaaua auucuauugc uauuccaaca aauuuuacca uuucugugac  2220 aacagaaauu uuaccugugu cuaugacaaa aacaucugug gauuguacca uguacauuug  2280 uggagauucu acagaauguu cuaaucugcu gcugcaguau ggaucuuuuu guacacagcu  2340 gaauagagcu uuaacaggaa uugcugugga acaggauaaa aauacacagg aaguguuugc  2400 ucaggugaaa cagauuuaca aaacaccacc aauuaaagau uuuggaggau uuaauuuuag  2460 ccagauucug ccugauccuu cuaaaccuuc uaaaagaucu uuuauugaag aucugcuguu  2520 uaauaaagug acacuggcag augcaggauu uauuaaacag uauggagauu gccuggguga  2580 uauugcugca agagaucuga uuugugcuca gaaauuuaau ggacugacag ugcugccucc  2640 ucugcugaca gaugaaauga uugcucagua cacaucugcu uuacuggcug gaacaauuac  2700 aagcggaugg acauuuggag cuggagcugc ucugcagauu ccuuuugcaa ugcagauggc  2760 uuacagauuu aauggaauug gagugacaca gaauguguua uaugaaaauc agaaacugau  2820 ugcaaaucag uuuaauucug caauuggcaa aauucaggau ucucugucuu cuacagcuuc  2880 ugcucuggga aaacugcagg auguggugaa ucagaaugca caggcacuga auacucuggu  2940 gaaacagcug ucuagcaauu uuggggcaau uucuucugug cugaaugaua uucugucuag  3000 acuggauccu ccugaagcug aagugcagau ugauagacug aucacaggaa gacugcaguc  3060 ucugcagacu uaugugacac agcagcugau uagagcugcu gaaauuagag cuucugcuaa  3120 ucuggcugcu acaaaaaugu cugaaugugu gcugggacag ucaaaaagag uggauuuuug  3180 uggaaaagga uaucaucuga ugucuuuucc acagucugcu ccacauggag ugguguuuuu  3240 acaugugaca uaugugccag cacaggaaaa gaauuuuacc acagcaccag caauuuguca  3300 ugauggaaaa gcacauuuuc caagagaagg aguguuugug ucuaauggaa cacauugguu  3360 ugugacacag agaaauuuuu augaaccuca gauuauuaca acagauaaua cauuuguguc  3420 aggaaauugu gaugugguga uuggaauugu gaauaauaca guguaugauc cacugcagcc  3480 agaacuggau ucuuuuaaag aagaacugga uaaauauuuu aaaaaucaca caucuccuga  3540 uguggauuua ggagauauuu cuggaaucaa ugcaucugug gugaauauuc agaaagaaau  3600 ugauagacug aaugaagugg ccaaaaaucu gaaugaaucu cugauugauc ugcaggaacu  3660 uggaaaauau gaacaguaca uuaaauggcc uugguacauu uggcuuggau uuauugcagg  3720 auuaauugca auugugaugg ugacaauuau guuauguugu augacaucau guuguucuug  3780 uuuaaaagga uguuguucuu guggaagcug uuguaaauuu gaugaagaug auucugaacc  3840 uguguuaaaa ggagugaaau ugcauuacac augaugacuc gagcugguac ugcaugcacg  3900 caaugcuagc ugccccuuuc ccguccuggg uaccccgagu cucccccgac cucggguccc  3960 agguaugcuc ccaccuccac cugccccacu caccaccucu gcuaguucca gacaccuccc  4020 aagcacgcag caaugcagcu caaaacgcuu agccuagcca cacccccacg ggaaacagca  4080 gugauuaacc uuuagcaaua aacgaaaguu uaacuaagcu auacuaaccc caggguuggu  4140 caauuucgug ccagccacac ccuggagcua gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa  4200 aagcauauga cuaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  4260 aaaaaaaaaa aaaaaaaaaa aa                                           4282 RBL063.2 (SEQ ID NO: 29 nucleotide; SEQ ID NO: 9 amino acid) Structure beta-S-ARCA(D1)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (S1S2 full-length protein, sequence variant) SEQ ID NO: 29 gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccauguucgu    60 guuccuggug cugcugccuc ugguguccag ccagugugug aaccugacca ccagaacaca   120 gcugccucca gccuacacca acagcuuuac cagaggcgug uacuaccccg acaagguguu   180 cagauccagc gugcugcacu cuacccagga ccuguuccug ccuuucuuca gcaacgugac   240 cugguuccac gccauccacg uguccggcac caauggcacc aagagauucg acaaccccgu   300 gcugcccuuc aacgacgggg uguacuuugc cagcaccgag aaguccaaca ucaucagagg   360 cuggaucuuc ggcaccacac uggacagcaa gacccagagc cugcugaucg ugaacaacgc   420 caccaacgug gucaucaaag ugugcgaguu ccaguucugc aacgaccccu uccugggcgu   480 cuacuaccac aagaacaaca agagcuggau ggaaagcgag uuccgggugu acagcagcgc   540 caacaacugc accuucgagu acguguccca gccuuuccug auggaccugg aaggcaagca   600 gggcaacuuc aagaaccugc gcgaguucgu guuuaagaac aucgacggcu acuucaagau   660 cuacagcaag cacaccccua ucaaccucgu gcgggaucug ccucagggcu ucucugcucu   720 ggaaccccug guggaucugc ccaucggcau caacaucacc cgguuucaga cacugcuggc   780 ccugcacaga agcuaccuga caccuggcga uagcagcagc ggauggacag cuggugccgc   840 cgcuuacuau gugggcuacc ugcagccuag aaccuuccug cugaaguaca acgagaacgg   900 caccaucacc gacgccgugg auugugcucu ggauccucug agcgagacaa agugcacccu   960 gaaguccuuc accguggaaa agggcaucua ccagaccagc aacuuccggg ugcagcccac  1020 cgaauccauc gugcgguucc ccaauaucac caaucugugc cccuucggcg agguguucaa  1080 ugccaccaga uucgccucug uguacgccug gaaccggaag cggaucagca auugcguggc  1140 cgacuacucc gugcuguaca acuccgccag cuucagcacc uucaagugcu acggcguguc  1200 cccuaccaag cugaacgacc ugugcuucac aaacguguac gccgacagcu ucgugauccg  1260 gggagaugaa gugcggcaga uugccccugg acagacaggc aagaucgccg acuacaacua  1320 caagcugccc gacgacuuca ccggcugugu gauugccugg aacagcaaca accuggacuc  1380 caaagucggc ggcaacuaca auuaccugua ccggcuguuc cggaagucca aucugaagcc  1440 cuucgagcgg gacaucucca ccgagaucua ucaggccggc agcaccccuu guaacggcgu  1500 ggaaggcuuc aacugcuacu ucccacugca guccuacggc uuucagccca caaauggcgu  1560 gggcuaucag cccuacagag ugguggugcu gagcuucgaa cugcugcaug ccccugccac  1620 agugugcggc ccuaagaaaa gcaccaaucu cgugaagaac aaaugcguga acuucaacuu  1680 caacggccug accggcaccg gcgugcugac agagagcaac aagaaguucc ugccauucca  1740 gcaguuuggc cgggauaucg ccgauaccac agacgccguu agagaucccc agacacugga  1800 aauccuggac aucaccccuu gcagcuucgg cggagugucu gugaucaccc cuggcaccaa  1860 caccagcaau cagguggcag ugcuguacca ggacgugaac uguaccgaag ugcccguggc  1920 cauucacgcc gaucagcuga caccuacaug gcggguguac uccaccggca gcaauguguu  1980 ucagaccaga gccggcuguc ugaucggagc cgagcacgug aacaauagcu acgagugcga  2040 cauccccauc ggcgcuggaa ucugcgccag cuaccagaca cagacaaaca gcccucggag  2100 agccagaagc guggccagcc agagcaucau ugccuacaca augucucugg gcgccgagaa  2160 cagcguggcc uacuccaaca acucuaucgc uauccccacc aacuucacca ucagcgugac  2220 cacagagauc cugccugugu ccaugaccaa gaccagcgug gacugcacca uguacaucug  2280 cggcgauucc accgagugcu ccaaccugcu gcugcaguac ggcagcuucu gcacccagcu  2340 gaauagagcc cugacaggga ucgccgugga acaggacaag aacacccaag agguguucgc  2400 ccaagugaag cagaucuaca agaccccucc uaucaaggac uucggcggcu ucaauuucag  2460 ccagauucug cccgauccua gcaagcccag caagcggagc uucaucgagg accugcuguu  2520 caacaaagug acacuggccg acgccggcuu caucaagcag uauggcgauu gucugggcga  2580 cauugccgcc agggaucuga uuugcgccca gaaguuuaac ggacugacag ugcugccucc  2640 ucugcugacc gaugagauga ucgcccagua cacaucugcc cugcuggccg gcacaaucac  2700 aagcggcugg acauuuggag caggcgccgc ucugcagauc cccuuugcua ugcagauggc  2760 cuaccgguuc aacggcaucg gagugaccca gaaugugcug uacgagaacc agaagcugau  2820 cgccaaccag uucaacagcg ccaucggcaa gauccaggac agccugagca gcacagcaag  2880 cgcccuggga aagcugcagg acguggucaa ccagaaugcc caggcacuga acacccuggu  2940 caagcagcug uccuccaacu ucggcgccau cagcucugug cugaacgaua uccugagcag  3000 acuggacccu ccugaggccg aggugcagau cgacagacug aucacaggca gacugcagag  3060 ccuccagaca uacgugaccc agcagcugau cagagccgcc gagauuagag ccucugccaa  3120 ucuggccgcc accaagaugu cugagugugu gcugggccag agcaagagag uggacuuuug  3180 cggcaagggc uaccaccuga ugagcuuccc ucagucugcc ccucacggcg ugguguuucu  3240 gcacgugaca uaugugcccg cucaagagaa gaauuucacc accgcuccag ccaucugcca  3300 cgacggcaaa gcccacuuuc cuagagaagg cguguucgug uccaacggca cccauugguu  3360 cgugacacag cggaacuucu acgagcccca gaucaucacc accgacaaca ccuucguguc  3420 uggcaacugc gacgucguga ucggcauugu gaacaauacc guguacgacc cucugcagcc  3480 cgagcuggac agcuucaaag aggaacugga caaguacuuu aagaaccaca caagccccga  3540 cguggaccug ggcgauauca gcggaaucaa ugccagcguc gugaacaucc agaaagagau  3600 cgaccggcug aacgaggugg ccaagaaucu gaacgagagc cugaucgacc ugcaagaacu  3660 ggggaaguac gagcaguaca ucaaguggcc cugguacauc uggcugggcu uuaucgccgg  3720 acugauugcc aucgugaugg ucacaaucau gcuguguugc augaccagcu gcuguagcug  3780 ccugaagggc uguuguagcu guggcagcug cugcaaguuc gacgaggacg auucugagcc  3840 cgugcugaag ggcgugaaac ugcacuacac augaugacuc gagcugguac ugcaugcacg  3900 caaugcuagc ugccccuuuc ccguccuggg uaccccgagu cucccccgac cucggguccc  3960 agguaugcuc ccaccuccac cugccccacu caccaccucu gcuaguucca gacaccuccc  4020 aagcacgcag caaugcagcu caaaacgcuu agccuagcca cacccccacg ggaaacagca  4080 gugauuaacc uuuagcaaua aacgaaaguu uaacuaagcu auacuaaccc caggguuggu  4140 caauuucgug ccagccacac ccuggagcua gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa  4200 aagcauauga cuaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  4260 aaaaaaaaaa aaaaaaaaaa aa                                           4282 BNT162a1; RBL063.3 (SEQ ID NO: 30 nucleotide; SEQ ID NO: 21 amino acid) Structure beta-S-ARCA(D1)-hAg-Kozak-RBD-GS-Fibritin-FI-A30L70 Encoded antigen Viral spike protein (S protein) of the SARS-CoV-2 (partial sequence,Receptor Binding Domain (RBD)  of S1S2 protein) SEQ ID NO: 30 gggcgaacua guauucuucu gguccccaca gacucagaga gaacccgcca ccauguuugu    60 guuucuugug cugcugccuc uugugucuuc ucagugugug gugagauuuc caaauauuac   120 aaaucugugu ccauuuggag aaguguuuaa ugcaacaaga uuugcaucug uguaugcaug   180 gaauagaaaa agaauuucua auuguguggc ugauuauucu gugcuguaua auagugcuuc   240 uuuuuccaca uuuaaauguu auggaguguc uccaacaaaa uuaaaugauu uauguuuuac   300 aaauguguau gcugauucuu uugugaucag aggugaugaa gugagacaga uugcccccgg   360 acagacagga aaaauugcug auuacaauua caaacugccu gaugauuuua caggaugugu   420 gauugcuugg aauucuaaua auuuagauuc uaaaguggga ggaaauuaca auuaucugua   480 cagacuguuu agaaaaucaa aucugaaacc uuuugaaaga gauauuucaa cagaaauuua   540 ucaggcugga ucaacaccuu guaauggagu ggaaggauuu aauuguuauu uuccauuaca   600 gagcuaugga uuucagccaa ccaauggugu gggauaucag ccauauagag ugguggugcu   660 gucuuuugaa cugcugcaug caccugcaac agugugugga ccuaaaggcu cccccggcuc   720 cggcuccgga ucugguuaua uuccugaagc uccaagagau gggcaagcuu acguucguaa   780 agauggcgaa uggguauuac uuucuaccuu uuuaggccgg ucccuggagg ugcuguucca   840 gggccccggc ugaugacucg agcugguacu gcaugcacgc aaugcuagcu gccccuuucc   900 cguccugggu accccgaguc ucccccgacc ucggguccca gguaugcucc caccuccacc   960 ugccccacuc accaccucug cuaguuccag acaccuccca agcacgcagc aaugcagcuc  1020 aaaacgcuua gccuagccac acccccacgg gaaacagcag ugauuaaccu uuagcaauaa  1080 acgaaaguuu aacuaagcua uacuaacccc aggguugguc aauuucgugc cagccacacc  1140 cuggagcuag caaaaaaaaa aaaaaaaaaa aaaaaaaaaa agcauaugac uaaaaaaaaa  1200 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  1260 a                                                                  1261 BNT162b2; RBP020.1 (SEQ ID NO: 31 nucleotide; SEQ ID NO: 9 amino acid) Structure m₂ ^(7,3′-O)-Gppp(m₁ ^(2′-O))ApG)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (S1S2 full-length protein, sequence variant) SEQ ID NO: 31 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguuug    60 uguuucuugu gcugcugccu cuugugucuu cucagugugu gaauuugaca acaagaacac   120 agcugccacc agcuuauaca aauucuuuua ccagaggagu guauuauccu gauaaagugu   180 uuagaucuuc ugugcugcac agcacacagg accuguuucu gccauuuuuu agcaauguga   240 caugguuuca ugcaauucau gugucuggaa caaauggaac aaaaagauuu gauaauccug   300 ugcugccuuu uaaugaugga guguauuuug cuucaacaga aaagucaaau auuauuagag   360 gauggauuuu uggaacaaca cuggauucua aaacacaguc ucugcugauu gugaauaaug   420 caacaaaugu ggugauuaaa gugugugaau uucaguuuug uaaugauccu uuucugggag   480 uguauuauca caaaaauaau aaaucuugga uggaaucuga auuuagagug uauuccucug   540 caaauaauug uacauuugaa uaugugucuc agccuuuucu gauggaucug gaaggaaaac   600 agggcaauuu uaaaaaucug agagaauuug uguuuaaaaa uauugaugga uauuuuaaaa   660 uuuauucuaa acacacacca auuaauuuag ugagagaucu gccucaggga uuuucugcuc   720 uggaaccucu gguggaucug ccaauuggca uuaauauuac aagauuucag acacugcugg   780 cucugcacag aucuuaucug acaccuggag auucuucuuc uggauggaca gccggagcug   840 cagcuuauua ugugggcuau cugcagccaa gaacauuucu gcugaaauau aaugaaaaug   900 gaacaauuac agaugcugug gauugugcuc uggauccucu gucugaaaca aaauguacau   960 uaaaaucuuu uacaguggaa aaaggcauuu aucagacauc uaauuuuaga gugcagccaa  1020 cagaaucuau ugugagauuu ccaaauauua caaaucugug uccauuugga gaaguguuua  1080 augcaacaag auuugcaucu guguaugcau ggaauagaaa aagaauuucu aauugugugg  1140 cugauuauuc ugugcuguau aauagugcuu cuuuuuccac auuuaaaugu uauggagugu  1200 cuccaacaaa auuaaaugau uuauguuuua caaaugugua ugcugauucu uuugugauca  1260 gaggugauga agugagacag auugcccccg gacagacagg aaaaauugcu gauuacaauu  1320 acaaacugcc ugaugauuuu acaggaugug ugauugcuug gaauucuaau aauuuagauu  1380 cuaaaguggg aggaaauuac aauuaucugu acagacuguu uagaaaauca aaucugaaac  1440 cuuuugaaag agauauuuca acagaaauuu aucaggcugg aucaacaccu uguaauggag  1500 uggaaggauu uaauuguuau uuuccauuac agagcuaugg auuucagcca accaauggug  1560 ugggauauca gccauauaga gugguggugc ugucuuuuga acugcugcau gcaccugcaa  1620 cagugugugg accuaaaaaa ucuacaaauu uagugaaaaa uaaaugugug aauuuuaauu  1680 uuaauggauu aacaggaaca ggagugcuga cagaaucuaa uaaaaaauuu cugccuuuuc  1740 agcaguuugg cagagauauu gcagauacca cagaugcagu gagagauccu cagacauuag  1800 aaauucugga uauuacaccu uguucuuuug gggguguguc ugugauuaca ccuggaacaa  1860 auacaucuaa ucagguggcu gugcuguauc aggaugugaa uuguacagaa gugccagugg  1920 caauucaugc agaucagcug acaccaacau ggagagugua uucuacagga ucuaaugugu  1980 uucagacaag agcaggaugu cugauuggag cagaacaugu gaauaauucu uaugaaugug  2040 auauuccaau uggagcaggc auuugugcau cuuaucagac acagacaaau uccccaagga  2100 gagcaagauc uguggcaucu cagucuauua uugcauacac caugucucug ggagcagaaa  2160 auucuguggc auauucuaau aauucuauug cuauuccaac aaauuuuacc auuucuguga  2220 caacagaaau uuuaccugug ucuaugacaa aaacaucugu ggauuguacc auguacauuu  2280 guggagauuc uacagaaugu ucuaaucugc ugcugcagua uggaucuuuu uguacacagc  2340 ugaauagagc uuuaacagga auugcugugg aacaggauaa aaauacacag gaaguguuug  2400 cucaggugaa acagauuuac aaaacaccac caauuaaaga uuuuggagga uuuaauuuua  2460 gccagauucu gccugauccu ucuaaaccuu cuaaaagauc uuuuauugaa gaucugcugu  2520 uuaauaaagu gacacuggca gaugcaggau uuauuaaaca guauggagau ugccugggug  2580 auauugcugc aagagaucug auuugugcuc agaaauuuaa uggacugaca gugcugccuc  2640 cucugcugac agaugaaaug auugcucagu acacaucugc uuuacuggcu ggaacaauua  2700 caagcggaug gacauuugga gcuggagcug cucugcagau uccuuuugca augcagaugg  2760 cuuacagauu uaauggaauu ggagugacac agaauguguu auaugaaaau cagaaacuga  2820 uugcaaauca guuuaauucu gcaauuggca aaauucagga uucucugucu ucuacagcuu  2880 cugcucuggg aaaacugcag gaugugguga aucagaaugc acaggcacug aauacucugg  2940 ugaaacagcu gucuagcaau uuuggggcaa uuucuucugu gcugaaugau auucugucua  3000 gacuggaucc uccugaagcu gaagugcaga uugauagacu gaucacagga agacugcagu  3060 cucugcagac uuaugugaca cagcagcuga uuagagcugc ugaaauuaga gcuucugcua  3120 aucuggcugc uacaaaaaug ucugaaugug ugcugggaca gucaaaaaga guggauuuuu  3180 guggaaaagg auaucaucug augucuuuuc cacagucugc uccacaugga gugguguuuu  3240 uacaugugac auaugugcca gcacaggaaa agaauuuuac cacagcacca gcaauuuguc  3300 augauggaaa agcacauuuu ccaagagaag gaguguuugu gucuaaugga acacauuggu  3360 uugugacaca gagaaauuuu uaugaaccuc agauuauuac aacagauaau acauuugugu  3420 caggaaauug ugauguggug auuggaauug ugaauaauac aguguaugau ccacugcagc  3480 cagaacugga uucuuuuaaa gaagaacugg auaaauauuu uaaaaaucac acaucuccug  3540 auguggauuu aggagauauu ucuggaauca augcaucugu ggugaauauu cagaaagaaa  3600 uugauagacu gaaugaagug gccaaaaauc ugaaugaauc ucugauugau cugcaggaac  3660 uuggaaaaua ugaacaguac auuaaauggc cuugguacau uuggcuugga uuuauugcag  3720 gauuaauugc aauugugaug gugacaauua uguuauguug uaugacauca uguuguucuu  3780 guuuaaaagg auguuguucu uguggaagcu guuguaaauu ugaugaagau gauucugaac  3840 cuguguuaaa aggagugaaa uugcauuaca caugaugacu cgagcuggua cugcaugcac  3900 gcaaugcuag cugccccuuu cccguccugg guaccccgag ucucccccga ccucgggucc  3960 cagguaugcu cccaccucca ccugccccac ucaccaccuc ugcuaguucc agacaccucc  4020 caagcacgca gcaaugcagc ucaaaacgcu uagccuagcc acacccccac gggaaacagc  4080 agugauuaac cuuuagcaau aaacgaaagu uuaacuaagc uauacuaacc ccaggguugg  4140 ucaauuucgu gccagccaca cccuggagcu agcaaaaaaa aaaaaaaaaa aaaaaaaaaa  4200 aaagcauaug acuaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  4260 aaaaaaaaaa aaaaaaaaaa aaa                                          4283 RBP020.2 (SEQ ID NO: 10 nucleotide; SEQ ID NO: 9 amino acid) (see Table 1) Structure m₂ ^(7,3′-O)-Gppp(m₁ ^(2′-O))ApG)-hAg-Kozak-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (S1S2 full-length protein, sequence variant) BNT162b1; RBP020.3 (SEQ ID NO: 32; SEQ ID NO: 21 amino acid) Structure m₂ ^(7,3′-O)-Gppp(m₁ ^(2′-O))ApG)-hAg-Kozak-RBD-GS-Fibritin-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (partial sequence, SEQ ID NO: 32 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguuug    60 uguuucuugu gcugcugccu cuugugucuu cucagugugu ggugagauuu ccaaauauua   120 caaaucugug uccauuugga gaaguguuua augcaacaag auuugcaucu guguaugcau   180 ggaauagaaa aagaauuucu aauugugugg cugauuauuc ugugcuguau aauagugcuu   240 cuuuuuccac auuuaaaugu uauggagugu cuccaacaaa auuaaaugau uuauguuuua   300 caaaugugua ugcugauucu uuugugauca gaggugauga agugagacag auugcccccg   360 gacagacagg aaaaauugcu gauuacaauu acaaacugcc ugaugauuuu acaggaugug   420 ugauugcuug gaauucuaau aauuuagauu cuaaaguggg aggaaauuac aauuaucugu   480 acagacuguu uagaaaauca aaucugaaac cuuuugaaag agauauuuca acagaaauuu   540 aucaggcugg aucaacaccu uguaauggag uggaaggauu uaauuguuau uuuccauuac   600 agagcuaugg auuucagcca accaauggug ugggauauca gccauauaga gugguggugc   660 ugucuuuuga acugcugcau gcaccugcaa cagugugugg accuaaaggc ucccccggcu   720 ccggcuccgg aucugguuau auuccugaag cuccaagaga ugggcaagcu uacguucgua   780 aagauggcga auggguauua cuuucuaccu uuuuaggccg gucccuggag gugcuguucc   840 agggccccgg cugaugacuc gagcugguac ugcaugcacg caaugcuagc ugccccuuuc   900 ccguccuggg uaccccgagu cucccccgac cucggguccc agguaugcuc ccaccuccac   960 cugccccacu caccaccucu gcuaguucca gacaccuccc aagcacgcag caaugcagcu  1020 caaaacgcuu agccuagcca cacccccacg ggaaacagca gugauuaacc uuuagcaaua  1080 aacgaaaguu uaacuaagcu auacuaaccc caggguuggu caauuucgug ccagccacac  1140 ccuggagcua gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa aagcauauga cuaaaaaaaa  1200 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  1260 aa                                                                 1262 RBS004.1 (SEQ ID NO: 33; SEQ ID NO: 9 amino acid) Structure beta-S-ARCA(D1)-replicase-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S protein) of the SARS-CoV-2 (S1S2 full-length protein, sequence variant) SEQ ID NO: 33 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac    60 guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu   120 gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau   180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga   240 agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga   300 ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag   360 gaaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac   420 ccugaccugg aaacugagac uaugugccuc cacgacgacg agucgugucg cuacgaaggg   480 caagucgcug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa   540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu   600 aagaacuugg cuggagcaua uccaucauac ucuaccaacu gggccgacga aaccguguua   660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug   720 uccauucuua gaaagaagua uuugaaacca uccaacaaug uucuauucuc uguuggcucg   780 accaucuacc acgaaaagag ggacuuacug aggagcuggc accugccguc uguauuucac   840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac   900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu   960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg  1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua  1080 cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu  1140 auagucguca acggucgcac ccagagaaac accaauacca ugaaaaauua ccuuuugccc  1200 guaguggccc aggcauuugc uaggugggca aaggaauaua aggaagauca agaagaugaa  1260 aggccacuag gacuacgaga uagacaguua gucauggggu guuguugggc uuuuagaagg  1320 cacaagauaa caucuauuua uaagcgcccg gauacccaaa ccaucaucaa agugaacagc  1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaaca cauuggagau cgggcugaga  1440 acaagaauca ggaaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag  1500 gacguacaag aagcuaagug cgcagccgau gaggcuaagg aggugcguga agccgaggag  1560 uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agcccacucu ggaagccgau  1620 gucgacuuga uguuacaaga ggcuggggcc ggcucagugg agacaccucg uggcuugaua  1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag  1740 gcuguacuca agagugaaaa auuaucuugc auccacccuc ucgcugaaca agucauagug  1800 auaacacacu cuggccgaaa agggcguuau gccguggaac cauaccaugg uaaaguagug  1860 gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc  1920 auuguguaca acgaacguga guucguaaac agguaccugc accauauugc cacacaugga  1980 ggagcgcuga acacugauga agaauauuac aaaacuguca agcccagcga gcacgacggc  2040 gaauaccugu acgacaucga caggaaacag ugcgucaaga aagagcuagu cacugggcua  2100 gggcucacag gcgagcuggu cgauccuccc uuccaugaau ucgccuacga gagucugaga  2160 acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca  2220 ggcaagucug gcaucauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag  2280 aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau  2340 gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau  2400 auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga  2460 ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuuaa caugaugugc  2520 cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc  2580 cguugcacua aaucugugac uucggucguc ucaaccuugu uuuacgacaa aaaaaugaga  2640 acgacgaauc cgaaagagac uaagauugug auugacacua ccggcaguac caaaccuaag  2700 caggacgauc ucauucucac uuguuucaga ggguggguga agcaguugca aauagauuac  2760 aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau  2820 gccguucggu acaaggugaa ugaaaauccu cuguacgcac ccaccucaga acaugugaac  2880 guccuacuga cccgcacgga ggaccgcauc guguggaaaa cacuagccgg cgacccaugg  2940 auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa  3000 gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc  3060 cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc  3120 auagacauga ccacugaaca auggaacacu guggauuauu uugaaacgga caaagcucac  3180 ucagcagaga uaguauugaa ccaacuaugc gugagguucu uuggacucga ucuggacucc  3240 ggucuauuuu cugcacccac uguuccguua uccauuagga auaaucacug ggauaacucc  3300 ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac  3360 ccacaacugc cucgggcagu ugccacuggu agagucuaug acaugaacac ugguacacug  3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagacugcc ucaugcuuua  3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag  3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg  3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau  3660 gugcccaaau augacauaau auuuguuaau gugaggaccc cauauaaaua ccaucacuau  3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau  3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa  3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc  3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg  3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu auacagguuc cagacuccac  4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa  4080 ggagugauua uaaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg  4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga  4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu  4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc  4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg  4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau  4440 gcagauguag ccauauacug cagggacaag aaaugggaaa ugacucucaa ggaagcagug  4500 gcuaggagag aagcagugga ggagauaugc auauccgacg auucuucagu gacagaaccu  4560 gaugcagagc uggugagggu gcaucccaag aguucuuugg cuggaaggaa gggcuacagc  4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag  4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc  4740 auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaggagucg  4800 gaagccucca caccaccuag cacgcugccu ugcuugugca uccaugccau gacuccagaa  4860 agaguacagc gccuaaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca  4920 uugccgaagu auagaaucac uggugugcag aagauccaau gcucccagcc uauauuguuc  4980 ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua  5040 gacgagacuc cggagccauc ggcagagaac caauccacag aggggacacc ugaacaacca  5100 ccacuuauaa ccgaggauga gaccaggacu agaacgccug agccgaucau caucgaagaa  5160 gaagaagaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc  5220 gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca  5280 uccgacuuug auguggacag uuuauccaua cuugacaccc uggagggagc uagcgugacc  5340 agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg  5400 cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca  5460 agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg  5520 ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucacgcacu  5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug  5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu  5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa  5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc  5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu  5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu  5940 auucugcaag gccuagggca uuauuugaag gcagaaggaa aaguggagug cuaccgaacc  6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc  6060 gcaguggaag ccuguaacgc cauguugaaa gagaacuuuc cgacuguggc uucuuacugu  6120 auuauuccag aguacgaugc cuauuuggac augguugacg gagcuucaug cugcuuagac  6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacacuc cuauuuggaa  6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca  6300 gcugccacaa aaagaaauug caaugucacg caaaugagag aauugcccgu auuggauucg  6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg  6420 uuuaaagaaa accccaucag gcuuacugaa gaaaacgugg uaaauuacau uaccaaauua  6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac  6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca  6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca  6660 gcguaucugu gcggaaucca ccgagagcug guuaggagau uaaaugcggu ccugcuuccg  6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac  6780 uuccagccug gggauugugu ucuggaaacu gacaucgcgu cguuugauaa aagugaggac  6840 gacgccaugg cucugaccgc guuaaugauu cuggaagacu uaggugugga cgcagagcug  6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu  6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca  7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu  7080 gcagcauuca uuggagauga caauaucgug aaaggaguca aaucggacaa auuaauggca  7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag  7200 aaagcgccuu auuucugugg aggguuuauu uugugugacu ccgugaccgg cacagcgugc  7260 cguguggcag acccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau  7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug  7380 gguauucuuu cagagcugug caaggcagua gaaucaaggu augaaaccgu aggaacuucc  7440 aucauaguua uggccaugac uacucuagcu agcaguguua aaucauucag cuaccugaga  7500 ggggccccua uaacucucua cggcuaaccu gaauggacua cgacauaguc uaguccgcca  7560 agacuaguau guuuguguuu cuugugcugc ugccucuugu gucuucucag ugugugaauu  7620 ugacaacaag aacacagcug ccaccagcuu auacaaauuc uuuuaccaga ggaguguauu  7680 auccugauaa aguguuuaga ucuucugugc ugcacagcac acaggaccug uuucugccau  7740 uuuuuagcaa ugugacaugg uuucaugcaa uucauguguc uggaacaaau ggaacaaaaa  7800 gauuugauaa uccugugcug ccuuuuaaug auggagugua uuuugcuuca acagaaaagu  7860 caaauauuau uagaggaugg auuuuuggaa caacacugga uucuaaaaca cagucucugc  7920 ugauugugaa uaaugcaaca aaugugguga uuaaagugug ugaauuucag uuuuguaaug  7980 auccuuuucu gggaguguau uaucacaaaa auaauaaauc uuggauggaa ucugaauuua  8040 gaguguauuc cucugcaaau aauuguacau uugaauaugu gucucagccu uuucugaugg  8100 aucuggaagg aaaacagggc aauuuuaaaa aucugagaga auuuguguuu aaaaauauug  8160 auggauauuu uaaaauuuau ucuaaacaca caccaauuaa uuuagugaga gaucugccuc  8220 agggauuuuc ugcucuggaa ccucuggugg aucugccaau uggcauuaau auuacaagau  8280 uucagacacu gcuggcucug cacagaucuu aucugacacc uggagauucu ucuucuggau  8340 ggacagccgg agcugcagcu uauuaugugg gcuaucugca gccaagaaca uuucugcuga  8400 aauauaauga aaauggaaca auuacagaug cuguggauug ugcucuggau ccucugucug  8460 aaacaaaaug uacauuaaaa ucuuuuacag uggaaaaagg cauuuaucag acaucuaauu  8520 uuagagugca gccaacagaa ucuauuguga gauuuccaaa uauuacaaau cuguguccau  8580 uuggagaagu guuuaaugca acaagauuug caucugugua ugcauggaau agaaaaagaa  8640 uuucuaauug uguggcugau uauucugugc uguauaauag ugcuucuuuu uccacauuua  8700 aauguuaugg agugucucca acaaaauuaa augauuuaug uuuuacaaau guguaugcug  8760 auucuuuugu gaucagaggu gaugaaguga gacagauugc ccccggacag acaggaaaaa  8820 uugcugauua caauuacaaa cugccugaug auuuuacagg augugugauu gcuuggaauu  8880 cuaauaauuu agauucuaaa gugggaggaa auuacaauua ucuguacaga cuguuuagaa  8940 aaucaaaucu gaaaccuuuu gaaagagaua uuucaacaga aauuuaucag gcuggaucaa  9000 caccuuguaa uggaguggaa ggauuuaauu guuauuuucc auuacagagc uauggauuuc  9060 agccaaccaa ugguguggga uaucagccau auagaguggu ggugcugucu uuugaacugc  9120 ugcaugcacc ugcaacagug uguggaccua aaaaaucuac aaauuuagug aaaaauaaau  9180 gugugaauuu uaauuuuaau ggauuaacag gaacaggagu gcugacagaa ucuaauaaaa  9240 aauuucugcc uuuucagcag uuuggcagag auauugcaga uaccacagau gcagugagag  9300 auccucagac auuagaaauu cuggauauua caccuuguuc uuuugggggu gugucuguga  9360 uuacaccugg aacaaauaca ucuaaucagg uggcugugcu guaucaggau gugaauugua  9420 cagaagugcc aguggcaauu caugcagauc agcugacacc aacauggaga guguauucua  9480 caggaucuaa uguguuucag acaagagcag gaugucugau uggagcagaa caugugaaua  9540 auucuuauga augugauauu ccaauuggag caggcauuug ugcaucuuau cagacacaga  9600 caaauucccc aaggagagca agaucugugg caucucaguc uauuauugca uacaccaugu  9660 cucugggagc agaaaauucu guggcauauu cuaauaauuc uauugcuauu ccaacaaauu  9720 uuaccauuuc ugugacaaca gaaauuuuac cugugucuau gacaaaaaca ucuguggauu  9780 guaccaugua cauuugugga gauucuacag aauguucuaa ucugcugcug caguauggau  9840 cuuuuuguac acagcugaau agagcuuuaa caggaauugc uguggaacag gauaaaaaua  9900 cacaggaagu guuugcucag gugaaacaga uuuacaaaac accaccaauu aaagauuuug  9960 gaggauuuaa uuuuagccag auucugccug auccuucuaa accuucuaaa agaucuuuua 10020 uugaagaucu gcuguuuaau aaagugacac uggcagaugc aggauuuauu aaacaguaug 10080 gagauugccu gggugauauu gcugcaagag aucugauuug ugcucagaaa uuuaauggac 10140 ugacagugcu gccuccucug cugacagaug aaaugauugc ucaguacaca ucugcuuuac 10200 uggcuggaac aauuacaagc ggauggacau uuggagcugg agcugcucug cagauuccuu 10260 uugcaaugca gauggcuuac agauuuaaug gaauuggagu gacacagaau guguuauaug 10320 aaaaucagaa acugauugca aaucaguuua auucugcaau uggcaaaauu caggauucuc 10380 ugucuucuac agcuucugcu cugggaaaac ugcaggaugu ggugaaucag aaugcacagg 10440 cacugaauac ucuggugaaa cagcugucua gcaauuuugg ggcaauuucu ucugugcuga 10500 augauauucu gucuagacug gauccuccug aagcugaagu gcagauugau agacugauca 10560 caggaagacu gcagucucug cagacuuaug ugacacagca gcugauuaga gcugcugaaa 10620 uuagagcuuc ugcuaaucug gcugcuacaa aaaugucuga augugugcug ggacagucaa 10680 aaagagugga uuuuugugga aaaggauauc aucugauguc uuuuccacag ucugcuccac 10740 auggaguggu guuuuuacau gugacauaug ugccagcaca ggaaaagaau uuuaccacag 10800 caccagcaau uugucaugau ggaaaagcac auuuuccaag agaaggagug uuugugucua 10860 auggaacaca uugguuugug acacagagaa auuuuuauga accucagauu auuacaacag 10920 auaauacauu ugugucagga aauugugaug uggugauugg aauugugaau aauacagugu 10980 augauccacu gcagccagaa cuggauucuu uuaaagaaga acuggauaaa uauuuuaaaa 11040 aucacacauc uccugaugug gauuuaggag auauuucugg aaucaaugca ucugugguga 11100 auauucagaa agaaauugau agacugaaug aaguggccaa aaaucugaau gaaucucuga 11160 uugaucugca ggaacuugga aaauaugaac aguacauuaa auggccuugg uacauuuggc 11220 uuggauuuau ugcaggauua auugcaauug ugauggugac aauuauguua uguuguauga 11280 caucauguug uucuuguuua aaaggauguu guucuugugg aagcuguugu aaauuugaug 11340 aagaugauuc ugaaccugug uuaaaaggag ugaaauugca uuacacauga ugacucgagc 11400 ugguacugca ugcacgcaau gcuagcugcc ccuuucccgu ccuggguacc ccgagucucc 11460 cccgaccucg ggucccaggu augcucccac cuccaccugc cccacucacc accucugcua 11520 guuccagaca ccucccaagc acgcagcaau gcagcucaaa acgcuuagcc uagccacacc 11580 cccacgggaa acagcaguga uuaaccuuua gcaauaaacg aaaguuuaac uaagcuauac 11640 uaaccccagg guuggucaau uucgugccag ccacaccgcg gccgcaugaa uacagcagca 11700 auuggcaagc ugcuuacaua gaacucgcgg cgauuggcau gccgccuuaa aauuuuuauu 11760 uuauuuuuuc uuuucuuuuc cgaaucggau uuuguuuuua auauuucaaa aaaaaaaaaa 11820 aaaaaaaaaa aaaaaaagca uaugacuaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 11880 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa                          11917 RBS004.2 (SEQ ID NO: 34; SEQ ID NO: 9 amino acid) Structure beta-S-ARCA(D1)-replicase-S1S2-PP-FI-A30L70 Encoded antigen Viral spike protein (S protein) of the SARS-CoV-2 SEQ ID NO: 34 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac    60 guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu   120 gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau   180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga   240 agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga   300 ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag   360 gaaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac   420 ccugaccugg aaacugagac uaugugccuc cacgacgacg agucgugucg cuacgaaggg   480 caagucgcug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa   540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu   600 aagaacuugg cuggagcaua uccaucauac ucuaccaacu gggccgacga aaccguguua   660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug   720 uccauucuua gaaagaagua uuugaaacca uccaacaaug uucuauucuc uguuggcucg   780 accaucuacc acgaaaagag ggacuuacug aggagcuggc accugccguc uguauuucac   840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac   900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu   960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg  1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua  1080 cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu  1140 auagucguca acggucgcac ccagagaaac accaauacca ugaaaaauua ccuuuugccc  1200 guaguggccc aggcauuugc uaggugggca aaggaauaua aggaagauca agaagaugaa  1260 aggccacuag gacuacgaga uagacaguua gucauggggu guuguugggc uuuuagaagg  1320 cacaagauaa caucuauuua uaagcgcccg gauacccaaa ccaucaucaa agugaacagc  1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaaca cauuggagau cgggcugaga  1440 acaagaauca ggaaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag  1500 gacguacaag aagcuaagug cgcagccgau gaggcuaagg aggugcguga agccgaggag  1560 uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agcccacucu ggaagccgau  1620 gucgacuuga uguuacaaga ggcuggggcc ggcucagugg agacaccucg uggcuugaua  1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag  1740 gcuguacuca agagugaaaa auuaucuugc auccacccuc ucgcugaaca agucauagug  1800 auaacacacu cuggccgaaa agggcguuau gccguggaac cauaccaugg uaaaguagug  1860 gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc  1920 auuguguaca acgaacguga guucguaaac agguaccugc accauauugc cacacaugga  1980 ggagcgcuga acacugauga agaauauuac aaaacuguca agcccagcga gcacgacggc  2040 gaauaccugu acgacaucga caggaaacag ugcgucaaga aagagcuagu cacugggcua  2100 gggcucacag gcgagcuggu cgauccuccc uuccaugaau ucgccuacga gagucugaga  2160 acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca  2220 ggcaagucug gcaucauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag  2280 aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau  2340 gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau  2400 auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga  2460 ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuuaa caugaugugc  2520 cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc  2580 cguugcacua aaucugugac uucggucguc ucaaccuugu uuuacgacaa aaaaaugaga  2640 acgacgaauc cgaaagagac uaagauugug auugacacua ccggcaguac caaaccuaag  2700 caggacgauc ucauucucac uuguuucaga ggguggguga agcaguugca aauagauuac  2760 aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau  2820 gccguucggu acaaggugaa ugaaaauccu cuguacgcac ccaccucaga acaugugaac  2880 guccuacuga cccgcacgga ggaccgcauc guguggaaaa cacuagccgg cgacccaugg  2940 auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa  3000 gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc  3060 cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc  3120 auagacauga ccacugaaca auggaacacu guggauuauu uugaaacgga caaagcucac  3180 ucagcagaga uaguauugaa ccaacuaugc gugagguucu uuggacucga ucuggacucc  3240 ggucuauuuu cugcacccac uguuccguua uccauuagga auaaucacug ggauaacucc  3300 ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac  3360 ccacaacugc cucgggcagu ugccacuggu agagucuaug acaugaacac ugguacacug  3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagacugcc ucaugcuuua  3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag  3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg  3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau  3660 gugcccaaau augacauaau auuuguuaau gugaggaccc cauauaaaua ccaucacuau  3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau  3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa  3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc  3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg  3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu auacagguuc cagacuccac  4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa  4080 ggagugauua uaaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg  4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga  4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu  4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc  4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg  4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau  4440 gcagauguag ccauauacug cagggacaag aaaugggaaa ugacucucaa ggaagcagug  4500 gcuaggagag aagcagugga ggagauaugc auauccgacg auucuucagu gacagaaccu  4560 gaugcagagc uggugagggu gcaucccaag aguucuuugg cuggaaggaa gggcuacagc  4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag  4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc  4740 auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaggagucg  4800 gaagccucca caccaccuag cacgcugccu ugcuugugca uccaugccau gacuccagaa  4860 agaguacagc gccuaaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca  4920 uugccgaagu auagaaucac uggugugcag aagauccaau gcucccagcc uauauuguuc  4980 ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua  5040 gacgagacuc cggagccauc ggcagagaac caauccacag aggggacacc ugaacaacca  5100 ccacuuauaa ccgaggauga gaccaggacu agaacgccug agccgaucau caucgaagaa  5160 gaagaagaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc  5220 gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca  5280 uccgacuuug auguggacag uuuauccaua cuugacaccc uggagggagc uagcgugacc  5340 agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg  5400 cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca  5460 agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg  5520 ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucacgcacu  5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug  5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu  5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa  5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc  5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu  5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu  5940 auucugcaag gccuagggca uuauuugaag gcagaaggaa aaguggagug cuaccgaacc  6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc  6060 gcaguggaag ccuguaacgc cauguugaaa gagaacuuuc cgacuguggc uucuuacugu  6120 auuauuccag aguacgaugc cuauuuggac augguugacg gagcuucaug cugcuuagac  6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacacuc cuauuuggaa  6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca  6300 gcugccacaa aaagaaauug caaugucacg caaaugagag aauugcccgu auuggauucg  6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg  6420 uuuaaagaaa accccaucag gcuuacugaa gaaaacgugg uaaauuacau uaccaaauua  6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac  6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca  6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca  6660 gcguaucugu gcggaaucca ccgagagcug guuaggagau uaaaugcggu ccugcuuccg  6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac  6780 uuccagccug gggauugugu ucuggaaacu gacaucgcgu cguuugauaa aagugaggac  6840 gacgccaugg cucugaccgc guuaaugauu cuggaagacu uaggugugga cgcagagcug  6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu  6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca  7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu  7080 gcagcauuca uuggagauga caauaucgug aaaggaguca aaucggacaa auuaauggca  7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag  7200 aaagcgccuu auuucugugg aggguuuauu uugugugacu ccgugaccgg cacagcgugc  7260 cguguggcag acccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau  7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug  7380 gguauucuuu cagagcugug caaggcagua gaaucaaggu augaaaccgu aggaacuucc  7440 aucauaguua uggccaugac uacucuagcu agcaguguua aaucauucag cuaccugaga  7500 ggggccccua uaacucucua cggcuaaccu gaauggacua cgacauaguc uaguccgcca  7560 agacuaguau guucguguuc cuggugcugc ugccucuggu guccagccag ugugugaacc  7620 ugaccaccag aacacagcug ccuccagccu acaccaacag cuuuaccaga ggcguguacu  7680 accccgacaa gguguucaga uccagcgugc ugcacucuac ccaggaccug uuccugccuu  7740 ucuucagcaa cgugaccugg uuccacgcca uccacguguc cggcaccaau ggcaccaaga  7800 gauucgacaa ccccgugcug cccuucaacg acggggugua cuuugccagc accgagaagu  7860 ccaacaucau cagaggcugg aucuucggca ccacacugga cagcaagacc cagagccugc  7920 ugaucgugaa caacgccacc aacgugguca ucaaagugug cgaguuccag uucugcaacg  7980 accccuuccu gggcgucuac uaccacaaga acaacaagag cuggauggaa agcgaguucc  8040 ggguguacag cagcgccaac aacugcaccu ucgaguacgu gucccagccu uuccugaugg  8100 accuggaagg caagcagggc aacuucaaga accugcgcga guucguguuu aagaacaucg  8160 acggcuacuu caagaucuac agcaagcaca ccccuaucaa ccucgugcgg gaucugccuc  8220 agggcuucuc ugcucuggaa ccccuggugg aucugcccau cggcaucaac aucacccggu  8280 uucagacacu gcuggcccug cacagaagcu accugacacc uggcgauagc agcagcggau  8340 ggacagcugg ugccgccgcu uacuaugugg gcuaccugca gccuagaacc uuccugcuga  8400 aguacaacga gaacggcacc aucaccgacg ccguggauug ugcucuggau ccucugagcg  8460 agacaaagug cacccugaag uccuucaccg uggaaaaggg caucuaccag accagcaacu  8520 uccgggugca gcccaccgaa uccaucgugc gguuccccaa uaucaccaau cugugccccu  8580 ucggcgaggu guucaaugcc accagauucg ccucugugua cgccuggaac cggaagcgga  8640 ucagcaauug cguggccgac uacuccgugc uguacaacuc cgccagcuuc agcaccuuca  8700 agugcuacgg cguguccccu accaagcuga acgaccugug cuucacaaac guguacgccg  8760 acagcuucgu gauccgggga gaugaagugc ggcagauugc cccuggacag acaggcaaga  8820 ucgccgacua caacuacaag cugcccgacg acuucaccgg cugugugauu gccuggaaca  8880 gcaacaaccu ggacuccaaa gucggcggca acuacaauua ccuguaccgg cuguuccgga  8940 aguccaaucu gaagcccuuc gagcgggaca ucuccaccga gaucuaucag gccggcagca  9000 ccccuuguaa cggcguggaa ggcuucaacu gcuacuuccc acugcagucc uacggcuuuc  9060 agcccacaaa uggcgugggc uaucagcccu acagaguggu ggugcugagc uucgaacugc  9120 ugcaugcccc ugccacagug ugcggcccua agaaaagcac caaucucgug aagaacaaau  9180 gcgugaacuu caacuucaac ggccugaccg gcaccggcgu gcugacagag agcaacaaga  9240 aguuccugcc auuccagcag uuuggccggg auaucgccga uaccacagac gccguuagag  9300 auccccagac acuggaaauc cuggacauca ccccuugcag cuucggcgga gugucuguga  9360 ucaccccugg caccaacacc agcaaucagg uggcagugcu guaccaggac gugaacugua  9420 ccgaagugcc cguggccauu cacgccgauc agcugacacc uacauggcgg guguacucca  9480 ccggcagcaa uguguuucag accagagccg gcugucugau cggagccgag cacgugaaca  9540 auagcuacga gugcgacauc cccaucggcg cuggaaucug cgccagcuac cagacacaga  9600 caaacagccc ucggagagcc agaagcgugg ccagccagag caucauugcc uacacaaugu  9660 cucugggcgc cgagaacagc guggccuacu ccaacaacuc uaucgcuauc cccaccaacu  9720 ucaccaucag cgugaccaca gagauccugc cuguguccau gaccaagacc agcguggacu  9780 gcaccaugua caucugcggc gauuccaccg agugcuccaa ccugcugcug caguacggca  9840 gcuucugcac ccagcugaau agagcccuga cagggaucgc cguggaacag gacaagaaca  9900 cccaagaggu guucgcccaa gugaagcaga ucuacaagac cccuccuauc aaggacuucg  9960 gcggcuucaa uuucagccag auucugcccg auccuagcaa gcccagcaag cggagcuuca 10020 ucgaggaccu gcuguucaac aaagugacac uggccgacgc cggcuucauc aagcaguaug 10080 gcgauugucu gggcgacauu gccgccaggg aucugauuug cgcccagaag uuuaacggac 10140 ugacagugcu gccuccucug cugaccgaug agaugaucgc ccaguacaca ucugcccugc 10200 uggccggcac aaucacaagc ggcuggacau uuggagcagg cgccgcucug cagauccccu 10260 uugcuaugca gauggccuac cgguucaacg gcaucggagu gacccagaau gugcuguacg 10320 agaaccagaa gcugaucgcc aaccaguuca acagcgccau cggcaagauc caggacagcc 10380 ugagcagcac agcaagcgcc cugggaaagc ugcaggacgu ggucaaccag aaugcccagg 10440 cacugaacac ccuggucaag cagcuguccu ccaacuucgg cgccaucagc ucugugcuga 10500 acgauauccu gagcagacug gacccuccug aggccgaggu gcagaucgac agacugauca 10560 caggcagacu gcagagccuc cagacauacg ugacccagca gcugaucaga gccgccgaga 10620 uuagagccuc ugccaaucug gccgccacca agaugucuga gugugugcug ggccagagca 10680 agagagugga cuuuugcggc aagggcuacc accugaugag cuucccucag ucugccccuc 10740 acggcguggu guuucugcac gugacauaug ugcccgcuca agagaagaau uucaccaccg 10800 cuccagccau cugccacgac ggcaaagccc acuuuccuag agaaggcgug uucgugucca 10860 acggcaccca uugguucgug acacagcgga acuucuacga gccccagauc aucaccaccg 10920 acaacaccuu cgugucuggc aacugcgacg ucgugaucgg cauugugaac aauaccgugu 10980 acgacccucu gcagcccgag cuggacagcu ucaaagagga acuggacaag uacuuuaaga 11040 accacacaag ccccgacgug gaccugggcg auaucagcgg aaucaaugcc agcgucguga 11100 acauccagaa agagaucgac cggcugaacg agguggccaa gaaucugaac gagagccuga 11160 ucgaccugca agaacugggg aaguacgagc aguacaucaa guggcccugg uacaucuggc 11220 ugggcuuuau cgccggacug auugccaucg ugauggucac aaucaugcug uguugcauga 11280 ccagcugcug uagcugccug aagggcuguu guagcugugg cagcugcugc aaguucgacg 11340 aggacgauuc ugagcccgug cugaagggcg ugaaacugca cuacacauga ugacucgagc 11400 ugguacugca ugcacgcaau gcuagcugcc ccuuucccgu ccuggguacc ccgagucucc 11460 cccgaccucg ggucccaggu augcucccac cuccaccugc cccacucacc accucugcua 11520 guuccagaca ccucccaagc acgcagcaau gcagcucaaa acgcuuagcc uagccacacc 11580 cccacgggaa acagcaguga uuaaccuuua gcaauaaacg aaaguuuaac uaagcuauac 11640 uaaccccagg guuggucaau uucgugccag ccacaccgcg gccgcaugaa uacagcagca 11700 auuggcaagc ugcuuacaua gaacucgcgg cgauuggcau gccgccuuaa aauuuuuauu 11760 uuauuuuuuc uuuucuuuuc cgaaucggau uuuguuuuua auauuucaaa aaaaaaaaaa 11820 aaaaaaaaaa aaaaaaagca uaugacuaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 11880 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa                          11917 BNT162c1; RBS004.3 (SEQ ID NO: 35; SEQ ID NO: 21 amino acid) Structure beta-S-ARCA(D1)-replicase-RBD-GS-Fibritin-FI-A30L70 Encoded antigen Viral spike protein (S protein) of the SARS-CoV-2 (partial sequence, Receptor Binding Domain (RBD) of S1S2 protein) SEQ ID NO: 35 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac    60 guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu   120 gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau   180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga   240 agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga   300 ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag   360 gaaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac   420 ccugaccugg aaacugagac uaugugccuc cacgacgacg agucgugucg cuacgaaggg   480 caagucgcug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa   540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu   600 aagaacuugg cuggagcaua uccaucauac ucuaccaacu gggccgacga aaccguguua   660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug   720 uccauucuua gaaagaagua uuugaaacca uccaacaaug uucuauucuc uguuggcucg   780 accaucuacc acgaaaagag ggacuuacug aggagcuggc accugccguc uguauuucac   840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac   900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu   960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg  1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua  1080 cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu  1140 auagucguca acggucgcac ccagagaaac accaauacca ugaaaaauua ccuuuugccc  1200 guaguggccc aggcauuugc uaggugggca aaggaauaua aggaagauca agaagaugaa  1260 aggccacuag gacuacgaga uagacaguua gucauggggu guuguugggc uuuuagaagg  1320 cacaagauaa caucuauuua uaagcgcccg gauacccaaa ccaucaucaa agugaacagc  1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaaca cauuggagau cgggcugaga  1440 acaagaauca ggaaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag  1500 gacguacaag aagcuaagug cgcagccgau gaggcuaagg aggugcguga agccgaggag  1560 uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agcccacucu ggaagccgau  1620 gucgacuuga uguuacaaga ggcuggggcc ggcucagugg agacaccucg uggcuugaua  1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag  1740 gcuguacuca agagugaaaa auuaucuugc auccacccuc ucgcugaaca agucauagug  1800 auaacacacu cuggccgaaa agggcguuau gccguggaac cauaccaugg uaaaguagug  1860 gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc  1920 auuguguaca acgaacguga guucguaaac agguaccugc accauauugc cacacaugga  1980 ggagcgcuga acacugauga agaauauuac aaaacuguca agcccagcga gcacgacggc  2040 gaauaccugu acgacaucga caggaaacag ugcgucaaga aagagcuagu cacugggcua  2100 gggcucacag gcgagcuggu cgauccuccc uuccaugaau ucgccuacga gagucugaga  2160 acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca  2220 ggcaagucug gcaucauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag  2280 aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau  2340 gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau  2400 auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga  2460 ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuuaa caugaugugc  2520 cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc  2580 cguugcacua aaucugugac uucggucguc ucaaccuugu uuuacgacaa aaaaaugaga  2640 acgacgaauc cgaaagagac uaagauugug auugacacua ccggcaguac caaaccuaag  2700 caggacgauc ucauucucac uuguuucaga ggguggguga agcaguugca aauagauuac  2760 aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau  2820 gccguucggu acaaggugaa ugaaaauccu cuguacgcac ccaccucaga acaugugaac  2880 guccuacuga cccgcacgga ggaccgcauc guguggaaaa cacuagccgg cgacccaugg  2940 auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa  3000 gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc  3060 cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc  3120 auagacauga ccacugaaca auggaacacu guggauuauu uugaaacgga caaagcucac  3180 ucagcagaga uaguauugaa ccaacuaugc gugagguucu uuggacucga ucuggacucc  3240 ggucuauuuu cugcacccac uguuccguua uccauuagga auaaucacug ggauaacucc  3300 ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac  3360 ccacaacugc cucgggcagu ugccacuggu agagucuaug acaugaacac ugguacacug  3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagacugcc ucaugcuuua  3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag  3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg  3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau  3660 gugcccaaau augacauaau auuuguuaau gugaggaccc cauauaaaua ccaucacuau  3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau  3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa  3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc  3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg  3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu auacagguuc cagacuccac  4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa  4080 ggagugauua uaaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg  4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga  4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu  4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc  4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg  4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau  4440 gcagauguag ccauauacug cagggacaag aaaugggaaa ugacucucaa ggaagcagug  4500 gcuaggagag aagcagugga ggagauaugc auauccgacg auucuucagu gacagaaccu  4560 gaugcagagc uggugagggu gcaucccaag aguucuuugg cuggaaggaa gggcuacagc  4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag  4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc  4740 auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaggagucg  4800 gaagccucca caccaccuag cacgcugccu ugcuugugca uccaugccau gacuccagaa  4860 agaguacagc gccuaaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca  4920 uugccgaagu auagaaucac uggugugcag aagauccaau gcucccagcc uauauuguuc  4980 ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua  5040 gacgagacuc cggagccauc ggcagagaac caauccacag aggggacacc ugaacaacca  5100 ccacuuauaa ccgaggauga gaccaggacu agaacgccug agccgaucau caucgaagaa  5160 gaagaagaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc  5220 gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca  5280 uccgacuuug auguggacag uuuauccaua cuugacaccc uggagggagc uagcgugacc  5340 agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg  5400 cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca  5460 agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg  5520 ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucacgcacu  5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug  5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu  5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa  5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc  5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu  5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu  5940 auucugcaag gccuagggca uuauuugaag gcagaaggaa aaguggagug cuaccgaacc  6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc  6060 gcaguggaag ccuguaacgc cauguugaaa gagaacuuuc cgacuguggc uucuuacugu  6120 auuauuccag aguacgaugc cuauuuggac augguugacg gagcuucaug cugcuuagac  6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacacuc cuauuuggaa  6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca  6300 gcugccacaa aaagaaauug caaugucacg caaaugagag aauugcccgu auuggauucg  6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg  6420 uuuaaagaaa accccaucag gcuuacugaa gaaaacgugg uaaauuacau uaccaaauua  6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac  6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca  6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca  6660 gcguaucugu gcggaaucca ccgagagcug guuaggagau uaaaugcggu ccugcuuccg  6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac  6780 uuccagccug gggauugugu ucuggaaacu gacaucgcgu cguuugauaa aagugaggac  6840 gacgccaugg cucugaccgc guuaaugauu cuggaagacu uaggugugga cgcagagcug  6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu  6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca  7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu  7080 gcagcauuca uuggagauga caauaucgug aaaggaguca aaucggacaa auuaauggca  7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag  7200 aaagcgccuu auuucugugg aggguuuauu uugugugacu ccgugaccgg cacagcgugc  7260 cguguggcag acccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau  7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug  7380 gguauucuuu cagagcugug caaggcagua gaaucaaggu augaaaccgu aggaacuucc  7440 aucauaguua uggccaugac uacucuagcu agcaguguua aaucauucag cuaccugaga  7500 ggggccccua uaacucucua cggcuaaccu gaauggacua cgacauaguc uaguccgcca  7560 agacuaguau guuuguguuu cuugugcugc ugccucuugu gucuucucag ugugugguga  7620 gauuuccaaa uauuacaaau cuguguccau uuggagaagu guuuaaugca acaagauuug  7680 caucugugua ugcauggaau agaaaaagaa uuucuaauug uguggcugau uauucugugc  7740 uguauaauag ugcuucuuuu uccacauuua aauguuaugg agugucucca acaaaauuaa  7800 augauuuaug uuuuacaaau guguaugcug auucuuuugu gaucagaggu gaugaaguga  7860 gacagauugc ccccggacag acaggaaaaa uugcugauua caauuacaaa cugccugaug  7920 auuuuacagg augugugauu gcuuggaauu cuaauaauuu agauucuaaa gugggaggaa  7980 auuacaauua ucuguacaga cuguuuagaa aaucaaaucu gaaaccuuuu gaaagagaua  8040 uuucaacaga aauuuaucag gcuggaucaa caccuuguaa uggaguggaa ggauuuaauu  8100 guuauuuucc auuacagagc uauggauuuc agccaaccaa ugguguggga uaucagccau  8160 auagaguggu ggugcugucu uuugaacugc ugcaugcacc ugcaacagug uguggaccua  8220 aaggcucccc cggcuccggc uccggaucug guuauauucc ugaagcucca agagaugggc  8280 aagcuuacgu ucguaaagau ggcgaauggg uauuacuuuc uaccuuuuua ggccgguccc  8340 uggaggugcu guuccagggc cccggcugau gacucgagcu gguacugcau gcacgcaaug  8400 cuagcugccc cuuucccguc cuggguaccc cgagucuccc ccgaccucgg gucccaggua  8460 ugcucccacc uccaccugcc ccacucacca ccucugcuag uuccagacac cucccaagca  8520 cgcagcaaug cagcucaaaa cgcuuagccu agccacaccc ccacgggaaa cagcagugau  8580 uaaccuuuag caauaaacga aaguuuaacu aagcuauacu aaccccaggg uuggucaauu  8640 ucgugccagc cacaccgcgg ccgcaugaau acagcagcaa uuggcaagcu gcuuacauag  8700 aacucgcggc gauuggcaug ccgccuuaaa auuuuuauuu uauuuuuucu uuucuuuucc  8760 gaaucggauu uuguuuuuaa uauuucaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagcau  8820 augacuaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  8880 aaaaaaaaaa aaaaaa                                                  8896 RBS004.4 (SEQ ID NO: 36; SEQ ID NO: 37) Structure beta-S-ARCA(D1)-replicase-RBD-GS-Fibritin-TM-FI-A30L70 Encoded antigen Viral spike protein (S protein) of the SARS-CoV-2 (partial sequence, Receptor Binding Domain (RBD) of S1S2 protein) SEQ ID NO: 36 gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac    60 guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu   120 gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau   180 cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga   240 agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga   300 ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag   360 gaaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac   420 ccugaccugg aaacugagac uaugugccuc cacgacgacg agucgugucg cuacgaaggg   480 caagucgcug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa   540 gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu   600 aagaacuugg cuggagcaua uccaucauac ucuaccaacu gggccgacga aaccguguua   660 acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug   720 uccauucuua gaaagaagua uuugaaacca uccaacaaug uucuauucuc uguuggcucg   780 accaucuacc acgaaaagag ggacuuacug aggagcuggc accugccguc uguauuucac   840 uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac   900 gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu   960 acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg  1020 gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua  1080 cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu  1140 auagucguca acggucgcac ccagagaaac accaauacca ugaaaaauua ccuuuugccc  1200 guaguggccc aggcauuugc uaggugggca aaggaauaua aggaagauca agaagaugaa  1260 aggccacuag gacuacgaga uagacaguua gucauggggu guuguugggc uuuuagaagg  1320 cacaagauaa caucuauuua uaagcgcccg gauacccaaa ccaucaucaa agugaacagc  1380 gauuuccacu cauucgugcu gcccaggaua ggcaguaaca cauuggagau cgggcugaga  1440 acaagaauca ggaaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag  1500 gacguacaag aagcuaagug cgcagccgau gaggcuaagg aggugcguga agccgaggag  1560 uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agcccacucu ggaagccgau  1620 gucgacuuga uguuacaaga ggcuggggcc ggcucagugg agacaccucg uggcuugaua  1680 aagguuacca gcuacgcugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag  1740 gcuguacuca agagugaaaa auuaucuugc auccacccuc ucgcugaaca agucauagug  1800 auaacacacu cuggccgaaa agggcguuau gccguggaac cauaccaugg uaaaguagug  1860 gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc  1920 auuguguaca acgaacguga guucguaaac agguaccugc accauauugc cacacaugga  1980 ggagcgcuga acacugauga agaauauuac aaaacuguca agcccagcga gcacgacggc  2040 gaauaccugu acgacaucga caggaaacag ugcgucaaga aagagcuagu cacugggcua  2100 gggcucacag gcgagcuggu cgauccuccc uuccaugaau ucgccuacga gagucugaga  2160 acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca  2220 ggcaagucug gcaucauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag  2280 aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau  2340 gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau  2400 auugacgagg cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga  2460 ccuaaaaagg cagugcucug cggagauccc aaacagugcg guuuuuuuaa caugaugugc  2520 cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc  2580 cguugcacua aaucugugac uucggucguc ucaaccuugu uuuacgacaa aaaaaugaga  2640 acgacgaauc cgaaagagac uaagauugug auugacacua ccggcaguac caaaccuaag  2700 caggacgauc ucauucucac uuguuucaga ggguggguga agcaguugca aauagauuac  2760 aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau  2820 gccguucggu acaaggugaa ugaaaauccu cuguacgcac ccaccucaga acaugugaac  2880 guccuacuga cccgcacgga ggaccgcauc guguggaaaa cacuagccgg cgacccaugg  2940 auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa  3000 gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc  3060 cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc  3120 auagacauga ccacugaaca auggaacacu guggauuauu uugaaacgga caaagcucac  3180 ucagcagaga uaguauugaa ccaacuaugc gugagguucu uuggacucga ucuggacucc  3240 ggucuauuuu cugcacccac uguuccguua uccauuagga auaaucacug ggauaacucc  3300 ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac  3360 ccacaacugc cucgggcagu ugccacuggu agagucuaug acaugaacac ugguacacug  3420 cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagacugcc ucaugcuuua  3480 guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag  3540 ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg  3600 uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau  3660 gugcccaaau augacauaau auuuguuaau gugaggaccc cauauaaaua ccaucacuau  3720 cagcagugug aagaccaugc cauuaagcua agcauguuga ccaagaaagc augucugcau  3780 cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa  3840 agcaucauug gugcuauagc gcggcaguuc aaguuuuccc gaguaugcaa accgaaaucc  3900 ucacuugagg agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg  3960 cacaauccuu acaagcuauc aucaaccuug accaacauuu auacagguuc cagacuccac  4020 gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa  4080 ggagugauua uaaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg  4140 cuguauaaga aauucccgga aaguuucgau uuacagccga ucgaaguagg aaaagcgcga  4200 cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu  4260 ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc  4320 aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg  4380 aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau  4440 gcagauguag ccauauacug cagggacaag aaaugggaaa ugacucucaa ggaagcagug  4500 gcuaggagag aagcagugga ggagauaugc auauccgacg auucuucagu gacagaaccu  4560 gaugcagagc uggugagggu gcaucccaag aguucuuugg cuggaaggaa gggcuacagc  4620 acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag  4680 gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc  4740 auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaggagucg  4800 gaagccucca caccaccuag cacgcugccu ugcuugugca uccaugccau gacuccagaa  4860 agaguacagc gccuaaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca  4920 uugccgaagu auagaaucac uggugugcag aagauccaau gcucccagcc uauauuguuc  4980 ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua  5040 gacgagacuc cggagccauc ggcagagaac caauccacag aggggacacc ugaacaacca  5100 ccacuuauaa ccgaggauga gaccaggacu agaacgccug agccgaucau caucgaagaa  5160 gaagaagaag auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc  5220 gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca  5280 uccgacuuug auguggacag uuuauccaua cuugacaccc uggagggagc uagcgugacc  5340 agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg  5400 cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca  5460 agaacaccgu cacuugcacc cagcagggcc ugcuccagaa ccagccuagu uuccaccccg  5520 ccaggcguga auagggugau cacuagagag gagcucgaag cgcuuacccc gucacgcacu  5580 ccuagcaggu cggucuccag aaccagccug gucuccaacc cgccaggcgu aaauagggug  5640 auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu  5700 gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa  5760 acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc  5820 cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu  5880 aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu  5940 auucugcaag gccuagggca uuauuugaag gcagaaggaa aaguggagug cuaccgaacc  6000 cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc  6060 gcaguggaag ccuguaacgc cauguugaaa gagaacuuuc cgacuguggc uucuuacugu  6120 auuauuccag aguacgaugc cuauuuggac augguugacg gagcuucaug cugcuuagac  6180 acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacacuc cuauuuggaa  6240 cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca  6300 gcugccacaa aaagaaauug caaugucacg caaaugagag aauugcccgu auuggauucg  6360 gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg  6420 uuuaaagaaa accccaucag gcuuacugaa gaaaacgugg uaaauuacau uaccaaauua  6480 aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac  6540 auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca  6600 aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca  6660 gcguaucugu gcggaaucca ccgagagcug guuaggagau uaaaugcggu ccugcuuccg  6720 aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac  6780 uuccagccug gggauugugu ucuggaaacu gacaucgcgu cguuugauaa aagugaggac  6840 gacgccaugg cucugaccgc guuaaugauu cuggaagacu uaggugugga cgcagagcug  6900 uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu  6960 aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca  7020 gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu  7080 gcagcauuca uuggagauga caauaucgug aaaggaguca aaucggacaa auuaauggca  7140 gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag  7200 aaagcgccuu auuucugugg aggguuuauu uugugugacu ccgugaccgg cacagcgugc  7260 cguguggcag acccccuaaa aaggcuguuu aagcuaggca aaccucuggc agcagacgau  7320 gaacaugaug augacaggag aagggcauug caugaggagu caacacgcug gaaccgagug  7380 gguauucuuu cagagcugug caaggcagua gaaucaaggu augaaaccgu aggaacuucc  7440 aucauaguua uggccaugac uacucuagcu agcaguguua aaucauucag cuaccugaga  7500 ggggccccua uaacucucua cggcuaaccu gaauggacua cgacauaguc uaguccgcca  7560 agacuaguau guuuguguuu cuugugcugc ugccucuugu gucuucucag ugugugguga  7620 gauuuccaaa uauuacaaau cuguguccau uuggagaagu guuuaaugca acaagauuug  7680 caucugugua ugcauggaau agaaaaagaa uuucuaauug uguggcugau uauucugugc  7740 uguauaauag ugcuucuuuu uccacauuua aauguuaugg agugucucca acaaaauuaa  7800 augauuuaug uuuuacaaau guguaugcug auucuuuugu gaucagaggu gaugaaguga  7860 gacagauugc ccccggacag acaggaaaaa uugcugauua caauuacaaa cugccugaug  7920 auuuuacagg augugugauu gcuuggaauu cuaauaauuu agauucuaaa gugggaggaa  7980 auuacaauua ucuguacaga cuguuuagaa aaucaaaucu gaaaccuuuu gaaagagaua  8040 uuucaacaga aauuuaucag gcuggaucaa caccuuguaa uggaguggaa ggauuuaauu  8100 guuauuuucc auuacagagc uauggauuuc agccaaccaa ugguguggga uaucagccau  8160 auagaguggu ggugcugucu uuugaacugc ugcaugcacc ugcaacagug uguggaccua  8220 aaggcucccc cggcuccggc uccggaucug guuauauucc ugaagcucca agagaugggc  8280 aagcuuacgu ucguaaagau ggcgaauggg uauuacuuuc uaccuuuuua ggaagcggca  8340 gcggaucuga acaguacauu aaauggccuu gguacauuug gcuuggauuu auugcaggau  8400 uaauugcaau ugugauggug acaauuaugu uauguuguau gacaucaugu uguucuuguu  8460 uaaaaggaug uuguucuugu ggaagcuguu guaaauuuga ugaagaugau ucugaaccug  8520 uguuaaaagg agugaaauug cauuacacau gaugacucga gcugguacug caugcacgca  8580 augcuagcug ccccuuuccc guccugggua ccccgagucu cccccgaccu cgggucccag  8640 guaugcuccc accuccaccu gccccacuca ccaccucugc uaguuccaga caccucccaa  8700 gcacgcagca augcagcuca aaacgcuuag ccuagccaca cccccacggg aaacagcagu  8760 gauuaaccuu uagcaauaaa cgaaaguuua acuaagcuau acuaacccca ggguugguca  8820 auuucgugcc agccacaccg cggccgcaug aauacagcag caauuggcaa gcugcuuaca  8880 uagaacucgc ggcgauuggc augccgccuu aaaauuuuua uuuuauuuuu ucuuuucuuu  8940 uccgaaucgg auuuuguuuu uaauauuuca aaaaaaaaaa aaaaaaaaaa aaaaaaaaag  9000 cauaugacua aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  9060 aaaaaaaaaa aaaaaaaaa                                               9079 SEQ ID NO: 37 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1               5                   10                  15 Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe             20                  25                  30 Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile         35                  40                  45 Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe     50                  55                  60 Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu 65                  70                  75                  80 Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu                 85                  90                  95 Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn             100                 105                 110 Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser         115                 120                 125 Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg     130                 135                 140 Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr 145                 150                 155                 160 Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe                 165                 170                 175 Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly             180                 185                 190 Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu         195                 200                 205 His Ala Pro Ala Thr Val Cys Gly Pro Lys Gly Ser Pro Gly Ser Gly     210                 215                 220 Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr 225                 230                 235                 240 Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Ser                 245                 250                 255 Gly Ser Gly Ser Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu             260                 265                 270 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu         275                 280                 285 Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys    290                 295                 300 Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys 305                 310                 315                 320 Gly Val Lys Leu His Tyr Thr                 325 BNT162b3c (SEQ ID NO: 38; SEQ ID NO: 39) Structure m₂ ^(7,3′-O)-Gppp(m₁ ^(2′-O))ApG-hAg-Kozak-RBD-GS-Fibritin-GS-TM-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (partial sequence,Receptor Binding Domain (RBD) of S1S2 protein fused to Fibritin fused to Transmembrane Domain (TM) of S1S2 protein); intrinsic S1S2 protein secretory signal peptide (aa 1-19) at the N-terminus of the antigen sequence SEQ ID NO: 38 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1               5                   10                  15 Asn Leu Thr Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly             20                  25                  30 Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg         35                  40                  45 Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser     50                  55                  60 Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu 65                  70                  75                  80 Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg                 85                  90                  95 Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala             100                 105                 110 Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala         115                 120                 125 Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr     130                 135                 140 Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp 145                 150                 155                 160 Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val                 165                 170                 175 Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro             180                 185                 190 Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe         195                 200                 205 Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Gly Ser Pro    210                 215                 220 Gly Ser Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly 225                 230                 235                 240 Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe                 245                 250                 255 Leu Gly Ser Gly Ser Gly Ser Glu Gln Tyr Ile Lys Trp Pro Trp Tyr             260                 265                 270 Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr         275                 280                 285 Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys     290                 295                 300 Cys Ser Cys Gly Ser Cys Cys 305                 310 SEQ ID NO: 39 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauguuug    60 uguuucuugu gcugcugccu cuugugucuu cucagugugu gaauuugaca gugagauuuc   120 caaauauuac aaaucugugu ccauuuggag aaguguuuaa ugcaacaaga uuugcaucug   180 uguaugcaug gaauagaaaa agaauuucua auuguguggc ugauuauucu gugcuguaua   240 auagugcuuc uuuuuccaca uuuaaauguu auggaguguc uccaacaaaa uuaaaugauu   300 uauguuuuac aaauguguau gcugauucuu uugugaucag aggugaugaa gugagacaga   360 uugcccccgg acagacagga aaaauugcug auuacaauua caaacugccu gaugauuuua   420 caggaugugu gauugcuugg aauucuaaua auuuagauuc uaaaguggga ggaaauuaca   480 auuaucugua cagacuguuu agaaaaucaa aucugaaacc uuuugaaaga gauauuucaa   540 cagaaauuua ucaggcugga ucaacaccuu guaauggagu ggaaggauuu aauuguuauu   600 uuccauuaca gagcuaugga uuucagccaa ccaauggugu gggauaucag ccauauagag   660 ugguggugcu gucuuuugaa cugcugcaug caccugcaac agugugugga ccuaaaggcu   720 cccccggcuc cggcuccgga ucugguuaua uuccugaagc uccaagagau gggcaagcuu   780 acguucguaa agauggcgaa uggguauuac uuucuaccuu uuuaggaagc ggcagcggau   840 cugaacagua cauuaaaugg ccuugguaca uuuggcuugg auuuauugca ggauuaauug   900 caauugugau ggugacaauu auguuauguu guaugacauc auguuguucu uguuuaaaag   960 gauguuguuc uuguggaagc uguuguugau gacucgagcu gguacugcau gcacgcaaug  1020 cuagcugccc cuuucccguc cuggguaccc cgagucuccc ccgaccucgg gucccaggua  1080 ugcucccacc uccaccugcc ccacucacca ccucugcuag uuccagacac cucccaagca  1140 cgcagcaaug cagcucaaaa cgcuuagccu agccacaccc ccacgggaaa cagcagugau  1200 uaaccuuuag caauaaacga aaguuuaacu aagcuauacu aaccccaggg uuggucaauu  1260 ucgugccagc cacacccugg agcuagcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaagca  1320 uaugacuaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  1380 aaaaaaaaaa aaaaaaa                                                 1397 BNT162b3d (SEQ ID NO: 40; SEQ ID NO: 41) Structure m₂ ^(7,3′-O)-Gppp(m₁ ^(2′-O))ApG-hAg-Kozak-RBD-GS-Fibritin-GS-TM-FI-A30L70 Encoded antigen Viral spike protein (S1S2 protein) of the SARS-CoV-2 (partial sequence, Receptor Binding Domain (RBD) of S1S2 protein fused to Fibritin fused to Transmembrane Domain (TM) of S1S2 protein); immunoglobulin secretory signal peptide (aa 1-22) at the N- terminus of the antigen sequence SEQ ID NO: 40 Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly 1               5                   10                  15 Ala His Ser Gln Met Gln Val Arg Phe Pro Asn Ile Thr Asn Leu Cys             20                  25                  30 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala         35                  40                  45 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu     50                  55                  60 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 65                  70                  75                  80 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe                 85                  90                  95 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly             100                 105                 110 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys         115                 120                 125 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn     130                 135                 140 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 145                 150                 155                 160 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys                165                 170                 175 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly             180                 185                 190 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val         195                 200                 205 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys     210                 215                 220 Gly Ser Pro Gly Ser Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro 225                 230                 235                 240 Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu                 245                 250                 255 Ser Thr Phe Leu Gly Ser Gly Ser Gly Ser Glu Gln Tyr Ile Lys Trp             260                 265                 270 Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val         275                 280                 285 Met Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu     290                 295                 300 Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys 305                 310 SEQ ID NO: 41 agaauaaacu aguauucuuc ugguccccac agacucagag agaacccgcc accauggauu    60 ggauuuggag aauccuguuc cucgugggag ccgcuacagg agcccacucc cagaugcagg   120 ugagauuucc aaauauuaca aaucuguguc cauuuggaga aguguuuaau gcaacaagau   180 uugcaucugu guaugcaugg aauagaaaaa gaauuucuaa uuguguggcu gauuauucug   240 ugcuguauaa uagugcuucu uuuuccacau uuaaauguua uggagugucu ccaacaaaau   300 uaaaugauuu auguuuuaca aauguguaug cugauucuuu ugugaucaga ggugaugaag   360 ugagacagau ugcccccgga cagacaggaa aaauugcuga uuacaauuac aaacugccug   420 augauuuuac aggaugugug auugcuugga auucuaauaa uuuagauucu aaagugggag   480 gaaauuacaa uuaucuguac agacuguuua gaaaaucaaa ucugaaaccu uuugaaagag   540 auauuucaac agaaauuuau caggcuggau caacaccuug uaauggagug gaaggauuua   600 auuguuauuu uccauuacag agcuauggau uucagccaac caauggugug ggauaucagc   660 cauauagagu gguggugcug ucuuuugaac ugcugcaugc accugcaaca guguguggac   720 cuaaaggcuc ccccggcucc ggcuccggau cugguuauau uccugaagcu ccaagagaug   780 ggcaagcuua cguucguaaa gauggcgaau ggguauuacu uucuaccuuu uuaggaagcg   840 gcagcggauc ugaacaguac auuaaauggc cuugguacau uuggcuugga uuuauugcag   900 gauuaauugc aauugugaug gugacaauua uguuauguug uaugacauca uguuguucuu   960 guuuaaaagg auguuguucu uguggaagcu guuguugaug acucgagcug guacugcaug  1020 cacgcaaugc uagcugcccc uuucccgucc uggguacccc gagucucccc cgaccucggg  1080 ucccagguau gcucccaccu ccaccugccc cacucaccac cucugcuagu uccagacacc  1140 ucccaagcac gcagcaaugc agcucaaaac gcuuagccua gccacacccc cacgggaaac  1200 agcagugauu aaccuuuagc aauaaacgaa aguuuaacua agcuauacua accccagggu  1260 uggucaauuu cgugccagcc acacccugga gcuagcaaaa aaaaaaaaaa aaaaaaaaaa  1320 aaaaaagcau augacuaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa  1380 aaaaaaaaaa aaaaaaaaaa aaaaaa                                       1406

Nucleic Acid Containing Particles

Nucleic acids described herein such as RNA encoding a payload may be administered formulated as particles.

In the context of the present disclosure, the term “particle” relates to a structured entity formed by molecules or molecule complexes. In some embodiments, the term “particle” relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure dispersed in a medium. In some embodiments, a particle is a nucleic acid containing particle such as a particle comprising DNA, RNA or a mixture thereof.

Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acid are involved in particle formation. This results in complexation and spontaneous formation of nucleic acid particles. In some embodiments, a nucleic acid particle is a nanoparticle.

As used in the present disclosure, “nanoparticle” refers to a particle having an average diameter suitable for parenteral administration.

A “nucleic acid particle” can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may be formed from at least one cationic or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid or lipid-like material and/or the cationic polymer combine together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.

In some embodiments, particles described herein further comprise at least one lipid or lipid-like material other than a cationic or cationically ionizable lipid or lipid-like material, at least one polymer other than a cationic polymer, or a mixture thereof

In some embodiments, nucleic acid particles comprise more than one type of nucleic acid molecules, where the molecular parameters of the nucleic acid molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features. Nucleic acid particles described herein may have an average diameter that in some embodiments ranges from about 30 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 70 nm to about 600 nm, from about 90 nm to about 400 nm, or from about 100 nm to about 300 nm.

Nucleic acid particles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, the nucleic acid particles can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

With respect to RNA lipid particles, the N/P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the RNA. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N/P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, RNA is considered to be completely bound to nanoparticles.

Nucleic acid particles described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles.

The term “colloid” as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out. The insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers. The mixture may be termed a colloid or a colloidal suspension. Sometimes the term “colloid” only refers to the particles in the mixture and not the entire suspension.

For the preparation of colloids comprising at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted. The most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (i) lipids dissolution in organic solvents, (ii) drying of the resultant solution, and (iii) hydration of dried lipid (using various aqueous media).

In the film hydration method, lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion. Furthermore, an additional downsizing step may be included.

Reverse phase evaporation is an alternative method to the film hydration for preparing liposomal vesicles that involves formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that turns subsequently into a liposomal suspension.

The term “ethanol injection technique” refers to a process, in which an ethanol solution comprising lipids is rapidly injected into an aqueous solution through a needle. This action disperses the lipids throughout the solution and promotes lipid structure formation, for example lipid vesicle formation such as liposome formation. Generally, the RNA lipoplex particles described herein are obtainable by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersion is, in some embodiments, formed as follows: an ethanol solution comprising lipids, such as cationic lipids and additional lipids, is injected into an aqueous solution under stirring. In some embodiments, the RNA lipoplex particles described herein are obtainable without a step of extrusion.

The term “extruding” or “extrusion” refers to the creation of particles having a fixed, cross-sectional profile. In particular, it refers to the downsizing of a particle, whereby the particle is forced through filters with defined pores.

Other methods having organic solvent free characteristics may also be used according to the present disclosure for preparing a colloid.

LNPs typically comprise four components: ionizable cationic lipids, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer conjugated lipid such as polyethylene glycol (PEG)-lipids. Each component is responsible for payload protection, and enables effective intracellular delivery. LNPs may be prepared by mixing lipids dissolved in ethanol rapidly with nucleic acid in an aqueous buffer.

The term “average diameter” refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Zaverage with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Here “average diameter”, “diameter” or “size” for particles is used synonymously with this value of the Zaverage.

The “polydispersity index” is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the “average diameter”. Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of nanoparticles.

Different types of nucleic acid containing particles have been described previously to be suitable for delivery of nucleic acid in particulate form (e.g. Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acid physically protects nucleic acid from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.

The present disclosure describes particles comprising nucleic acid, at least one cationic or cationically ionizable lipid or lipid-like material, and/or at least one cationic polymer which associate with nucleic acid to form nucleic acid particles and compositions comprising such particles. The nucleic acid particles may comprise nucleic acid which is complexed in different forms by non-covalent interactions to the particle. The particles described herein are not viral particles, in particular infectious viral particles, i.e., they are not able to virally infect cells. Suitable cationic or cationically ionizable lipids or lipid-like materials and cationic polymers are those that form nucleic acid particles and are included by the term “particle forming components” or “particle forming agents”. The term “particle forming components” or “particle forming agents” relates to any components which associate with nucleic acid to form nucleic acid particles. Such components include any component which can be part of nucleic acid particles.

Some embodiments described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species such as RNA species, e.g., a) a nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least a fragment of a parental virus protein, wherein amino acid positions in the at least a fragment of a parental virus protein are modified to comprise amino acids found in the corresponding amino acid positions of one or more virus protein variants; and b) a nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least a fragment of a parental virus protein, wherein amino acid positions in the at least a fragment of a parental virus protein are modified to comprise amino acids found in the corresponding amino acid positions of one or more virus protein variants.

In a particulate formulation, it is possible that each nucleic acid species is separately formulated as an individual particulate formulation. In that case, each individual particulate formulation will comprise one nucleic acid species. The individual particulate formulations may be present as separate entities, e.g. in separate containers. Such formulations are obtainable by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) together with a particle-forming agent, thereby allowing the formation of particles. Respective particles will contain exclusively the specific nucleic acid species that is being provided when the particles are formed (individual particulate formulations).

In some embodiments, a composition such as a pharmaceutical composition comprises more than one individual particle formulation. Respective pharmaceutical compositions are referred to as mixed particulate formulations. Mixed particulate formulations according to the invention are obtainable by forming, separately, individual particulate formulations, as described above, followed by a step of mixing of the individual particulate formulations. By the step of mixing, a formulation comprising a mixed population of nucleic acid-containing particles is obtainable. Individual particulate populations may be together in one container, comprising a mixed population of individual particulate formulations.

Alternatively, it is possible that different nucleic acid species are formulated together as a combined particulate formulation. Such formulations are obtainable by providing a combined formulation (typically combined solution) of different RNA species together with a particle-forming agent, thereby allowing the formation of particles. As opposed to a mixed particulate formulation, a combined particulate formulation will typically comprise particles which comprise more than one RNA species. In a combined particulate composition different RNA species are typically present together in a single particle.

Cationic Polymeric Materials (e.g., Polymers)

Given their high degree of chemical flexibility, polymeric materials are commonly used for nanoparticle-based delivery. Typically, cationic materials are used to electrostatically condense the negatively charged nucleic acid into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic materials useful in some embodiments herein. In addition, some investigators have synthesized polymeric materials specifically for nucleic acid delivery. Poly(β-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. In some embodiments, such synthetic materials may be suitable for use as cationic materials herein.

A “polymeric material”, as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. In some embodiments, such repeat units can all be identical; alternatively, in some cases, there can be more than one type of repeat unit present within the polymeric material. In some cases, a polymeric material is biologically derived, e.g., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymeric material, for example targeting moieties such as those described herein.

Those skilled in the art are aware that, when more than one type of repeat unit is present within a polymer (or polymeric moiety), then the polymer (or polymeric moiety) is said to be a “copolymer.” In some embodiments, a polymer (or polymeric moiety) utilized in accordance with the present disclosure may be a copolymer. Repeat units forming the copolymer can be arranged in any fashion. For example, in some embodiments, repeat units can be arranged in a random order; alternatively or additionally, in some embodiments, repeat units may be arranged in an alternating order, or as a “block” copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

In certain embodiments, a polymeric material for use in accordance with the present disclosure is biocompatible. Biocompatible materials are those that typically do not result in significant cell death at moderate concentrations. In certain embodiments, a biocompatible material is biodegradable, i.e., is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.

In certain embodiments, a polymeric material may be or comprise protamine or polyalkyleneimine, in particular protamine.

As those skilled in the art are aware term “protamine” is often used to refer to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term “protamine” is often used to refer to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.

In some embodiments, the term “protamine” as used herein is refers to a protamine amino acid sequence obtained or derived from natural or biological sources, including fragments thereof and/or multimeric forms of said amino acid sequence or fragment thereof, as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.

In some embodiments, a polyalkyleneimine comprises polyethylenimine and/or polypropylenimine, preferably polyethyleneimine. In some embodiments, a preferred polyalkyleneimine is polyethyleneimine (PEI). In some embodiments, the average molecular weight of PEI is preferably 0.75-102 to 107 Da, preferably 1000 to 105 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.

Preferred according to certain embodiments of the disclosure is linear polyalkyleneimine such as linear polyethyleneimine (PEI).

Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those which are able to electrostatically bind nucleic acid. In some embodiments, cationic polymeric materials contemplated for use herein include any cationic polymeric materials with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

In some embodiments, particles described herein may comprise polymers other than cationic polymers, e.g., non-cationic polymeric materials and/or anionic polymeric materials.

Collectively, anionic and neutral polymeric materials are referred to herein as non-cationic polymeric materials.

Lipid and Lipid-Like Material

The terms “lipid” and “lipid-like material” are used herein to refer to molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). In some embodiments, hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.

As used herein, the term “amphiphilic” refers to a molecule having both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.

The term “lipid-like material”, “lipid-like compound” or “lipid-like molecule” relates to substances that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids. As used herein, the term “lipid” is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.

Specific examples of amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.

In certain embodiments, the amphiphilic compound is a lipid. The term “lipid” refers to a group of organic compounds that are characterized by being insoluble in water, but soluble in many organic solvents. Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits). Although the term “lipid” is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

Fatty acids, or fatty acid residues are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.

Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word “triacylglycerol” is sometimes used synonymously with “triglyceride”. In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.

The glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived “tails” by ester linkages and to one “head” group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannose-containing headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides. Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins.

Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

According to the disclosure, lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

Cationic or Cationically Ionizable Lipids or Lipid-Like Materials

In some embodiments, nucleic acid particles described and/or utilized in accordance with the present disclosure may comprise at least one cationic or cationically ionizable lipid or lipid-like material as particle forming agent. Cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid. In some embodiments, cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein can be associated with nucleic acid, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

As used herein, a “cationic lipid” or “cationic lipid-like material” refers to a lipid or lipid-like material having a net positive charge. Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.

In certain embodiments, a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.

For purposes of the present disclosure, such “cationically ionizable” lipids or lipid-like materials are comprised by the term “cationic lipid or lipid-like material” unless contradicted by the circumstances.

In some embodiments, a cationic or cationically ionizable lipid or lipid-like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated.

Examples of cationic lipids include, but are not limited to: ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propanes; 1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl) 8,8′-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), Di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200); or heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate (SM-102).

In some embodiments, a cationic lipid is or comprises heptadecan-9-yl 8-((2-hydroxyethyl) (6-oxo-6-(undecyloxy) hexyl) amino) octanoate (SM-102). In some embodiments, a cationic lipid is or comprises a cationic lipid shown in the structure below.

In some embodiments, a cationic lipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) which is also referred to as ALC-0315 herein.

In some embodiments, a cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of the total lipid present in the particle.

In some particular embodiments, a particle for use in accordance with the present disclosure includes ALC-0315, for example in a weight percent within a range of about 40-55 mol percent of total lipids.

Additional Lipids or Lipid-Like Materials

In some embodiments, particles described herein comprise (e.g., in addition to a cationic lipid such as ALC315), one or more lipids or lipid-like materials other than cationic or cationically ionizable lipids or lipid-like materials, e.g., non-cationic lipids or lipid-like materials (including non-cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials. Optimizing the formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable/cationic lipid or lipid-like material may enhance particle stability and efficacy of nucleic acid delivery.

An additional lipid or lipid-like material may be incorporated which may or may not affect the overall charge of the nucleic acid particles. In certain embodiments, the additional lipid or lipid-like material is a non-cationic lipid or lipid-like material. The non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids. As used herein, an “anionic lipid” refers to any lipid that is negatively charged at a selected pH. As used herein, a “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. In preferred embodiments, the additional lipid comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains.

In certain preferred embodiments, the additional lipid is DSPC or DSPC and cholesterol. In certain embodiments, the nucleic acid particles include both a cationic lipid and an additional lipid.

In some embodiments, particles described herein include a polymer conjugated lipid such as a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art. In some embodiments, a pegylated lipid is ALC-0159, also referred to herein as (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).

Without wishing to be bound by theory, the amount of the at least one cationic lipid compared to the amount of the at least one additional lipid may affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. Accordingly, in some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

In some embodiments, the non-cationic lipid, in particular neutral lipid, (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in the particle.

In some embodiments, particles for use in accordance with the present disclosure may include, for example, ALC-0315, DSPC, CHOL, and ALC-0159, for example, wherein ALC-0315 is at about 40 to 55 mol percent; DSPC is at about 5 to 15 mol percent; CHOL is at about 30 to 50 mol percent; and ALC-0159 is at about 1 to 10 mol percent.

Lipoplex Particles

In certain embodiments of the present disclosure, an RNA may be present in RNA lipoplex particles.

In the context of the present disclosure, the term “RNA lipoplex particle” relates to a particle that contains lipid, in particular cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In some embodiments, a RNA lipoplex particle is a nanoparticle.

In certain embodiments, the RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

RNA lipoplex particles described herein have an average diameter that in some embodiments ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In an embodiment, the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

In some embodiments, RNA lipoplex particles and/or compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration. In some embodiments, RNA lipoplex particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase. In some embodiments, the aqueous phase has an acidic pH. In some embodiments, the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM. Liposomes may be used for preparing RNA lipoplex particles by mixing the liposomes with RNA. In some embodiments, the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid. In some embodiments, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In some embodiments, the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol) and/or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In some embodiments, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE). In some embodiments, the liposomes and RNA lipoplex particles comprise 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE).

Spleen targeting RNA lipoplex particles are described in WO 2013/143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs. In some embodiments, after administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in antigen presenting cells, such as professional antigen presenting cells in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells. In some embodiments, the antigen presenting cells are dendritic cells and/or macrophages.

Lipid Nanoparticles (LNPs)

In some embodiments, nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs). The LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

In some embodiments, the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.

In some embodiments, an LNP comprises from 40 to 55 mol percent, from 40 to 50 mol percent, from 41 to 49 mol percent, from 41 to 48 mol percent, from 42 to 48 mol percent, from 43 to 48 mol percent, from 44 to 48 mol percent, from 45 to 48 mol percent, from 46 to 48 mol percent, from 47 to 48 mol percent, or from 47.2 to 47.8 mol percent of the cationic lipid. In some embodiments, the LNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9 or 48.0 mol percent of the cationic lipid.

In some embodiments, the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent. In some embodiments, the neutral lipid is present in a concentration of about 9.5, 10 or 10.5 mol percent.

In some embodiments, the steroid is present in a concentration ranging from 30 to 50 mol percent, from 35 to 45 mol percent or from 38 to 43 mol percent. In some embodiments, the steroid is present in a concentration of about 40, 41, 42, 43, 44, 45 or 46 mol percent.

In some embodiments, the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer conjugated lipid.

In some embodiments, the LNP comprises from 40 to 50 mol percent a cationic lipid; from 5 to 15 mol percent of a neutral lipid; from 35 to 45 mol percent of a steroid; from 1 to 10 mol percent of a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.

In some embodiments, the mol percent is determined based on total mol of lipid present in the lipid nanoparticle.

In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.

In some embodiments, the steroid is cholesterol.

In some embodiments, the polymer conjugated lipid is a pegylated lipid. In some embodiments, the pegylated lipid has the following structure:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R12 and R13 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In some embodiments, R12 and R13 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some embodiments, w has a mean value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R12 and R13 are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45.

In some embodiments, the pegylated lipid is DMG-PEG 2000, e.g., having the following structure:

In some embodiments, the cationic lipid component of the LNPs has the structure of Formula (III):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L1 or L2 is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)x-, —S—S—, —C(═O)S—, SC(═O)—, —NRaC(═O)—, —C(═O)NRa—, NRaC(═O)NRa—, —OC(═O)NRa— or —NRaC(═O)O—, and the other of L1 or L2 is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)x-, —S—S—, —C(═O)S—, SC(═O)—, —NRaC(═O)—, —C(═O)NRa—, NRaC(═O)NRa—, —OC(═O)NRa— or —NRaC(═O)O— or a direct bond; G₁ and G₂ are each independently unsubstituted C₁-C₁₂ alkylene or C₁-C₁₂ alkenylene; G₃ is C₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈ cycloalkenylene; Ra is H or C₁-C₁₂ alkyl; R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;

R3 is H, OR5, CN, —C(═O)OR4, —OC(═O)R4 or —NR5C(═O)R4;

R4 is C1-C12 alkyl; R5 is H or C1-C6 alkyl; and x is 0, 1 or 2. In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

wherein: A is a 3 to 8-membered cycloalkyl or cycloalkylene ring; R6 is, at each occurrence, independently H, OH or C₁-C₂₄ alkyl; n is an integer ranging from 1 to 15. In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB). In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (IIID):

wherein y and z are each independently integers ranging from 1 to 12. In any of the foregoing embodiments of Formula (III), one of L1 or L2 is —O(C═O)—. For example, in some embodiments each of L1 and L2 are —O(C═O)—. In some different embodiments of any of the foregoing, L1 and L2 are each independently —(C═O)O— or —O(C═O)—. For example, in some embodiments each of L1 and L2 is —(C═O)O—. In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6. In some of the foregoing embodiments of Formula (III), R6 is H. In other of the foregoing embodiments, R6 is C1-C24 alkyl. In other embodiments, R6 is OH. In some embodiments of Formula (III), G3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3 is linear C1-C24 alkylene or linear C1-C24 alkenylene. In some other foregoing embodiments of Formula (III), R1 or R2, or both, is C6-C24 alkenyl. For example, in some embodiments, R1 and R2 each, independently have the following structure:

wherein: R7a and R7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7b and a are each selected such that R1 and R2 each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12. In some of the foregoing embodiments of Formula (III), at least one occurrence of R7a is H. For example, in some embodiments, R7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7b is C1-C8 alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl. In different embodiments of Formula (III), R1 or R2, or both, has one of the following structures:

In some of the foregoing embodiments of Formula (III), R3 is OH, CN, —C(═O)OR4, —OC(═O)R4 or —NHC(═O)R4. In some embodiments, R4 is methyl or ethyl. In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in the table below.

Representative Compounds of Formula (III).

No. Structure III-1 

III-2 

III-3 

III-4 

III-5 

III-6 

III-7 

III-8 

III-9 

III-10

III-11

III-12

III-13

III-14

III-15

III-16

III-17

III-18

III-19

III-20

III-21

III-22

III-23

III-24

III-25

III-26

III-27

III-28

III-29

III-30

III-31

III-32

III-33

III-34

III-35

III-36

In some embodiments, an LNP comprises a lipid of Formula (III), RNA, a neutral lipid, a steroid and a pegylated lipid. In some embodiments, a lipid of Formula (III) is compound III-3. In some embodiments, a neutral lipid is DSPC. In some embodiments, a steroid is cholesterol. In some embodiments, a pegylated lipid is ALC-0159.

In some embodiments, the cationic lipid is present in the LNP in an amount from about 40 to about 50 mole percent. In some embodiments, the neutral lipid is present in the LNP in an amount from about 5 to about 15 mole percent. In some embodiments, the steroid is present in the LNP in an amount from about 35 to about 45 mole percent. In some embodiments, the pegylated lipid is present in the LNP in an amount from about 1 to about 10 mole percent.

In some embodiments, the LNP comprises compound III-3 in an amount from about 40 to about 50 mole percent, DSPC in an amount from about 5 to about 15 mole percent, cholesterol in an amount from about 35 to about 45 mole percent, and ALC-0159 in an amount from about 1 to about 10 mole percent.

In some embodiments, the LNP comprises compound III-3 in an amount of about 47.5 mole percent, DSPC in an amount of about 10 mole percent, cholesterol in an amount of about 40.7 mole percent, and ALC-0159 in an amount of about 1.8 mole percent. In various different embodiments, the cationic lipid has one of the structures set forth in the table below.

No. Structure A

B

C

D

E

F

In some embodiments, the LNP comprises a cationic lipid shown in the above table, e.g., a cationic lipid of Formula (B) or Formula (D), in particular a cationic lipid of Formula (D), RNA, a neutral lipid, a steroid and a pegylated lipid. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the pegylated lipid is DMG-PEG 2000.

In some embodiments, the LNP comprises a cationic lipid that is an ionizable lipid-like material (lipidoid). In some embodiments, the cationic lipid has the following structure:

The N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N/P value is about 6.

LNP described herein may have an average diameter that in some embodiments ranges from about 30 nm to about 200 nm, or from about 60 nm to about 120 nm.

Pharmaceutical Compositions

In some embodiments, a pharmaceutical composition comprises an RNA polynucleotide disclosed herein formulated as a particle. In some embodiments, a particle is or comprises a lipid nanoparticle (LNP) or a lipoplex (LPX) particle.

In some embodiments, an RNA polynucleotide disclosed herein may be administered in a pharmaceutical composition or a medicament and may be administered in the form of any suitable pharmaceutical composition.

In some embodiments, a pharmaceutical composition described herein is an immunogenic composition for inducing an immune response. For example, in some embodiments, an immunogenic composition is a vaccine.

In some embodiments, an RNA polynucleotide disclosed herein may be administered in a pharmaceutical composition which may comprise a pharmaceutically acceptable carrier and may optionally comprise one or more adjuvants, stabilizers etc. In some embodiments, a pharmaceutical composition is for therapeutic or prophylactic treatments.

The term “adjuvant” relates to a compound which prolongs, enhances or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. The cytokines may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-a. Further known adjuvants are aluminium hydroxide, Freund's adjuvant or oil such as Montanide® ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys.

The pharmaceutical compositions according to the present disclosure are generally applied in a “pharmaceutically effective amount” and in “a pharmaceutically acceptable preparation”. The term “pharmaceutically acceptable” refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition. The term “pharmaceutically effective amount” or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

In some embodiments, a pharmaceutical composition disclosed herein may contain salts, buffers, preservatives, and optionally other therapeutic agents. In some embodiments, a pharmaceutical composition disclosed herein comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.

Suitable preservatives for use in a pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.

The term “excipient” as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.

The term “diluent” relates a diluting and/or thinning agent. Moreover, the term “diluent” includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol, glycerol and water. The term “carrier” refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carrier include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers. In some embodiments, the pharmaceutical composition of the present disclosure includes isotonic saline.

Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

In some embodiments, a pharmaceutical composition described herein may be administered intravenously, intraarterially, subcutaneously, intradermally or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, “parenteral administration” refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for intramuscular administration. In another embodiment, the pharmaceutical composition is formulated for systemic administration, e.g., for intravenous administration.

Characterization

In some embodiments, an RNA polynucleotide disclosed herein is characterized in that, when assessed in an organism administered a composition or medical preparation comprising an RNA polynucleotide, elevated expression of a payload is observed relative to an appropriate reference comparator.

In some embodiments, an RNA polynucleotide disclosed herein is characterized in that, when assessed in an organism administered a composition or medical preparation comprising an RNA polynucleotide, increased duration of expression (e.g., prolonged expression) of a payload is observed relative to an appropriate reference comparator.

In some embodiments, an RNA polynucleotide disclosed herein is characterized in that, when assessed in an organism administered a composition or medical preparation comprising an RNA polynucleotide, decreased interaction with IFIT1 of an RNA polynucleotide is observed relative to an appropriate reference comparator.

In some embodiments, an RNA polynucleotide disclosed herein is characterized in that, when assessed in an organism administered a composition or medical preparation comprising an RNA polynucleotide, increased translation an RNA polynucleotide is observed relative to an appropriate reference comparator.

In some embodiments, a reference comparator comprises an organism administered an otherwise similar RNA polynucleotide without a m7(3′OMeG)(5′)ppp(5′)(2′OMeA₁)pG₂ cap. In some embodiments, a reference comparator comprises an organism administered an otherwise similar RNA polynucleotide without a cap proximal sequence disclosed herein. In some embodiments, a reference comparator comprises an organism administered an otherwise similar RNA polynucleotide with a self-hybridizing sequence.

In some embodiments, an RNA polynucleotide disclosed herein is characterized in that, when assessed in an organism administered a composition or medical preparation comprising an RNA polynucleotide, elevated expression and increased duration of expression (e.g., prolonged expression) of a payload is observed relative to an appropriate reference comparator.

In some embodiments, elevated expression is determined at least 24 hours, at least 48 hours at least 72 hours, at least 96 hours or at least 120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression is determined at least 24 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression is determined at least 48 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression is determined at least 72 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression is determined at least 96 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression is determined at least 120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide.

In some embodiments, elevated expression is determined at about 24-120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression is determined at about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40-120 hours, about 50-120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours, or about 110-120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide.

In some embodiments, elevated expression of a payload is at least 2-fold to at least 10-fold. In some embodiments, elevated expression of a payload is at least 2-fold. In some embodiments, elevated expression of a payload is at least 3-fold. In some embodiments, elevated expression of a payload is at least 4-fold. In some embodiments, elevated expression of a payload is at least 6-fold. In some embodiments, elevated expression of a payload is at least 8-fold. In some embodiments, elevated expression of a payload is at least 10-fold.

In some embodiments, elevated expression of a payload is about 2-fold to about 50-fold. In some embodiments, elevated expression of a payload is about 2-fold to about 45-fold, about 2-fold to about 40-fold, about 2-fold to about 30-fold, about 2-fold to about 25-fold, about 2-fold to about 20-fold, about 2-fold to about 15-fold, about 2-fold to about 10-fold, about 2-fold to about 8-fold, about 2-fold to about 5-fold, about 5-fold to about 50-fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, about 20-fold to about 50-fold, about 25-fold to about 50-fold, about 30-fold to about 50-fold, about 40-fold to about 50-fold, or about 45-fold to about 50-fold.

In some embodiments, elevated expression (e.g., increased duration of expression) of a payload persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration of a composition or a medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression of a payload persists for at least 24 hours after administration. In some embodiments, elevated expression of a payload persists for at least 48 hours after administration. In some embodiments, elevated expression of a payload persists for at least 72 hours after administration. In some embodiments, elevated expression of a payload persists for at least 96 hours after administration. In some embodiments, elevated expression of a payload persists for at least 120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide.

In some embodiments, elevated expression of a payload persists for at about 24-120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide. In some embodiments, elevated expression persists for about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40-120 hours, about 50-120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours, or about 110-120 hours after administration of a composition or medical preparation comprising an RNA polynucleotide.

Uses

Disclosed herein, among other things, are methods of making and methods of using an RNA polynucleotide comprising a 5′cap; a 5′ UTR comprising a cap proximal structure; and a sequence encoding a payload.

In some embodiments, disclosed herein is an in vitro transcription reaction comprising: (i) a template DNA comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence disclosed herein; (ii) a polymerase; and (iii) an RNA polynucleotide. In some embodiments, a polymerase is or comprises a T7 polymerase. In some embodiments, a reaction further comprises a 5′ cap or a 5′ cap analog. In some embodiments, a 5′ cap analog is or comprises a Cap1 structure. In some embodiments, an RNA polynucleotide comprises a cap comprising a Cap1 structure; and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload. In some embodiments, a Cap1 structure comprises m7G(5′)ppp(5′)(2′OMeN1)pN2, wherein N1 is position +1 of the RNA polynucleotide, and N2 is position +2 of the RNA polynucleotide, and wherein N1 and N2 are each independently chosen from: A, C, G, or U.

In some embodiments, also disclosed herein is a method for producing a capped RNA comprising, transcribing a nucleic acid template in the presence of a cap structure, wherein the cap structure comprises G*ppp(m₁ ^(2-O))N₁pN₂,

wherein N₁ is complementary to position +1 of the nucleic acid template and N₂ is complementary to position +2 of the nucleic acid template, and N₁ and N₂ are independently chosen from A, C, G or U, wherein the RNA comprises: N₃ which is complementary to position +3 of the nucleic acid template and is any nucleotide, preferably A or C; N₄ which is complementary to position +4 of the nucleic acid template and is a nucleotide selected from the group consisting of A, G and U, preferably T; and N₅ which is complementary to position +5 of the nucleic acid template and is any nucleotide, wherein G* comprises the following structure:

wherein

represents the bond by which G* is bound to the first phosphor atom of the ppp group, R¹ is CH₃, R² is OH or O—CH₃, and R³ is O—CH₃.

In some embodiments, disclosed herein is a method of producing a polypeptide comprising a step of: providing an RNA polynucleotide that comprises a 5′ cap, a cap proximal sequence that comprises positions +1, +2, +3, +4, and +5 of an RNA polynucleotide, and a sequence encoding a payload; wherein an RNA polynucleotide is characterized in that when assessed in an organism administered an RNA polynucleotide or a composition comprising the same, elevated expression and/or increased duration of expression of an payload is observed relative to an appropriate reference comparator.

In some embodiments, disclosed herein is a method comprising: administering to a subject, a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle disclosed herein.

In some embodiments, disclosed herein is a method of inducing an immune response in a subject, comprising administering to a subject, a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle disclosed herein.

In some embodiments, disclosed herein is a method of vaccination of a subject, comprising administering to a subject, a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle disclosed herein.

In some embodiments, provided herein is a method of decreasing interaction with IFIT1 of an RNA polynucleotide that comprises a 5′ cap and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, the method comprising a step of: providing a variant of an RNA polynucleotide that differs from a parental RNA polynucleotide by substitution of one or more residues within the cap proximal sequence, and determining that interaction of a variant with IFIT1 is decreased relative to that of a parental RNA polynucleotide. In some embodiments, determining comprises administering the RNA polynucleotide or a composition comprising the same to a cell or an organism.

In some embodiments, disclosed herein is a method of increasing translatability of an RNA polynucleotide that comprises a 5′ cap, a cap proximal sequence that comprises positions +1, +2, +3, +4, and +5 of the RNA polynucleotide and a sequence encoding a payload, the method comprising a step of: providing a variant of an RNA polynucleotide that differs from a parental RNA polynucleotide by substitution of one or more residues within a cap proximal sequence; and determining that expression of a variant is increased relative to that of a parental RNA polynucleotide. In some embodiments, determining comprises administering the RNA polynucleotide or a composition comprising the same to a cell or an organism. In some embodiments, increased translatability is assessed by increased expression and/or a persistence of expression of the payload. In some embodiments, increased expression is determined at least 6 hours, at least 24 hours, at least 48 hours at least 72 hours, at least 96 hours or at least 120 hours after administering. In some embodiments, increase in expression is at least 2-fold to 10-fold. In some embodiments, increase in expression is about 2-fold to 50-fold. In some embodiments, elevated expression persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration.

In some embodiments, provide herein is a therapeutic RNA comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises: including one or more of the following residues in a cap proximal sequence: X at position +1 of an RNA polynucleotide, X at position +2 of an RNA polynucleotide, A at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and X at position +5 of an RNA polynucleotide, demonstrated to increase expression of the RNA when administered to a subject in an LNP formulation. In some embodiments, X is chosen from A, C, G or U.

In some embodiments, provide herein is a therapeutic RNA comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises: including one or more of the following residues in a cap proximal sequence: A at position +1 of an RNA polynucleotide, G at position +2 of an RNA polynucleotide, A at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and U at position +5 of an RNA polynucleotide, demonstrated to increase expression of the RNA when administered to a subject in an LNP formulation.

In some embodiments, provide herein is a therapeutic RNA comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises: including one or more of the following residues in a cap proximal sequence: X at position +1 of an RNA polynucleotide, X at position +2 of an RNA polynucleotide, C at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and X at position +5 of an RNA polynucleotide, demonstrated to increase expression of the RNA when administered to a subject in an LNP formulation. In some embodiments, X is chosen from A, C, G or U.

In some embodiments, provided herein is a method of increasing translation of an RNA polynucleotide comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises: including one or more of the following residues in a cap proximal sequence: X at position +1 of an RNA polynucleotide, X at position +2 of an RNA polynucleotide, A at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and X at position +5 of an RNA polynucleotide. In some embodiments, X is chosen from A, C, G or U.

In some embodiments, provided herein is a method of increasing translation of an RNA polynucleotide comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises: including one or more of the following residues in a cap proximal sequence: A at position +1 of an RNA polynucleotide, G at position +2 of an RNA polynucleotide, A at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and U at position +5 of an RNA polynucleotide.

In some embodiments, provided herein is a method of increasing translation of an RNA polynucleotide comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises: including one or more of the following residues in a cap proximal sequence: X at position +1 of an RNA polynucleotide, X at position +2 of an RNA polynucleotide, C at position +3 of an RNA polynucleotide, A at position +4 of an RNA polynucleotide, and X at position +5 of an RNA polynucleotide. In some embodiments, X is chosen from A, C, G or U.

In some embodiments of any of the methods disclosed herein, an immune response is induced in a subject. In some embodiments of any of the methods disclosed herein, an immune response is a prophylactic immune response or a therapeutic immune response.

In some embodiments of any of the methods disclosed herein, a subject is a mammal.

In some embodiments of any of the methods disclosed herein, a subject is a human.

In some embodiments of any of the methods disclosed herein, a subject has a disease or disorder disclosed herein.

In some embodiments of any of the methods disclosed herein, vaccination generates an immune response to an agent. In some embodiments, an immune response is a prophylactic immune response.

In some embodiments of any of the methods disclosed herein, a subject has a disease or disorder disclosed herein.

In some embodiments of any of the methods disclosed herein, one dose of a pharmaceutical composition is administered.

In some embodiments of any of the methods disclosed herein, a plurality of doses of a pharmaceutical composition is administered.

In some embodiments of any of the methods disclosed herein, the method further comprises administration of one or more therapeutic agents. In some embodiments, one or more therapeutic agents are administered before, after, or concurrently with administration of a pharmaceutical composition comprising an RNA polynucleotide.

Also provided herein in some embodiments, is a method of providing a framework for an RNA polynucleotide that comprises a 5′ cap, a cap proximal sequence, and a payload sequence, the method comprising a step of:

assessing at least two variants of an RNA polynucleotide, wherein:

each variant includes a same 5′ cap and payload sequence; and

the variants differ from one another at one or more specific residues of a cap proximal sequence;

wherein the assessing comprises determining expression levels and/or duration of expression of a payload; and selecting at least one combination of 5′ cap and a cap proximal sequence that displays elevated expression relative to at least one other combination.

In some embodiments, assessing comprises administering an RNA construct or a composition comprising the same to a cell or an organism:

In some embodiments, elevated expression of a payload is detected at a time point at least 6 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administering. In some embodiments, elevated expression is at least 2-fold to 10-fold. In some embodiments, elevated expression is about 2-fold to about 50-fold.

In some embodiments, elevated expression of a payload persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administering.

In some embodiments of any of the methods disclosed herein, an RNA polynucleotide comprises one or more features of an RNA polynucleotide provided herein.

In some embodiments of any of the methods disclosed herein, a composition comprising an RNA polynucleotide comprises a pharmaceutical composition provided herein.

ENUMERATED EMBODIMENTS

1. A composition or medical preparation comprising an RNA polynucleotide comprising:

a 5′ cap comprising a Cap1 structure; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein:

-   -   (i) the Cap1 structure comprises m7G(5′)ppp(5′)(2′OMeN₁)pN₂,         wherein N₁ is position +1 of the RNA polynucleotide, and N₂ is         position +2 of the RNA polynucleotide, and     -   wherein N₁ and N₂ are each independently chosen from: A, C, G,         or U; and     -   (ii) the cap proximal sequence comprises N₁ and N₂ of the Cap1         structure, and:         -   (a) a sequence selected from the group consisting of: A₃A₄X₅             (SEQ ID NO: 1); C₃A₄X₅ (SEQ ID NO: 2); A₃C₄A₅ (SEQ ID NO: 3)             and A₃U₄G₅ (SEQ ID NO: 4); or         -   (b) a sequence comprising: X₃Y₄X₅ (SEQ ID NO: 7);         -   wherein X₃ (nucleotide X at position +3 in SEQ ID NO: 7) or             X₅ (nucleotide X at position +5 in SEQ ID NO: 1 or SEQ ID             NO: 2) is each independently chosen from A, G, C, or U; and         -   wherein Y₄ (nucleotide Y at position +4 in SEQ ID NO: 7) is             not C.             2. The composition or medical preparation of embodiment 1,             wherein:     -   (i) the N₁ position is A and the N₂ position is G, and the Cap1         structure comprises m7G(5′)ppp(5′)(2′OMeA₁)pG₂;     -   (ii) the N₁ position is A and the N₂ position is U, and the Cap1         structure comprises m7G(5′)ppp(5′)(2′OMeA₁)pU₂; or     -   (iii) the N₁ position is G and the N₂ position is G, and the         Cap1 structure comprises m7G(5′)ppp(5′)(2′OMeG₁)pG₂.

3. The composition or medical preparation of embodiment 1 or 2, wherein a methylated Guanosine (m7G) in the Cap1 structure further comprises one or more modifications, e.g., wherein the m7G in the Cap1 structure comprises a methylated ribose.

4. The composition or medical preparation of embodiment 3, wherein the m7G in the Cap1 comprises a 3′O methylation (m7(3′OMeG)).

5. A composition or medical preparation comprising an RNA polynucleotide comprising: a 5′ cap; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of an RNA polynucleotide; and a sequence encoding a payload, wherein:

-   -   (i) the 5′ cap comprises a Cap1 structure comprising G*ppp(m₁         ^(2-O))N₁pN₂, wherein:         -   N₁ is position +1 of the RNA polynucleotide, and N₂ is             position +2 of the RNA polynucleotide, and wherein N₁ and N₂             are each independently chosen from: A, C, G, or U; and         -   G* comprises the following structure:

-   -   -   wherein             represents the bond by which G* is bound to the first             phosphor atom of the ppp group, R¹ is CH₃, R² is OH or             O—CH₃, and R³ is O—CH₃; and

    -   (ii) a cap proximal sequence comprises N₁ and N₂ of the Cap1         structure, and:         -   (a) a sequence chosen from the group consisting of: A₃A₄X₅             (SEQ ID NO: 1); C₃A₄X₅ (SEQ ID NO: 2); A₃C₄A₅ (SEQ ID NO: 3)             or A₃U₄G₅ (SEQ ID NO: 4); or         -   (b) a sequence comprising X₃Y₄X₅ (SEQ ID NO: 7);         -   wherein X₃ (nucleotide X at position +3 in SEQ ID NO: 7) or             X₅ (nucleotide X at position +5 in SEQ ID NO: 1 or SEQ ID             NO: 2) is each independently chosen from A, G, C, or U; and         -   wherein Y₄ (nucleotide Y at position +4 in SEQ ID NO: 7) is             not C.             6. The composition or medical preparation of embodiment 5,             wherein:

    -   (i) the N₁ position is A and the N₂ position is G, and the Cap1         structure comprises m7G(5′)ppp(5′)(2′OMeA₁)pG₂;

    -   (ii) the N₁ position is A and the N₂ position is U, and the Cap1         structure comprises m7G(5′)ppp(5′)(2′OMeA₁)pU₂; or

    -   (iii) the N₁ position is G and the N₂ position is G, and the         Cap1 structure comprises m7G(5′)ppp(5′)(2′OMeG₁)pG₂.         7. The composition or medical preparation of any one of the         preceding embodiments, wherein the cap proximal sequence         comprises N₁ and N₂ of the Cap1 structure, and a sequence         comprising: A₃A₄X₅ (SEQ ID NO: 1), optionally wherein X₅ is any         nucleotide, e.g., A, C, G or U, preferably U.         8. The composition or medical preparation of any one of         embodiments 1-6, wherein the cap proximal sequence comprises N₁         and N₂ of the Cap1 structure, and a sequence comprising C₃A₄X₅         (SEQ ID NO: 2), optionally wherein X₅ is any nucleotide, e.g.,         A, C, G or U.         9. The composition or medical preparation of any one of         embodiments 1-6, wherein the cap proximal sequence comprises N₁         and N₂ of the Cap1 structure, and a sequence comprising); X₃Y₄X₅         (SEQ ID NO: 7), wherein X₃ or X₅ are any nucleotide, e.g., A, G,         C, or U, and Y₄ is not C.         10. The composition or medical preparation of any one of         embodiments 1-6, wherein the cap proximal sequence comprises N₁         and N₂ of the Cap1 structure, and a sequence comprising A₃C₄A₅         (SEQ ID NO: 3).         11. The composition or medical preparation of any one of         embodiments 1-6, wherein the cap proximal sequence comprises N₁         and N₂ of the Cap1 structure, and a sequence comprising A₃U₄G₅         (SEQ ID NO: 4).         12. A composition or medical preparation comprising an RNA         polynucleotide comprising:

a 5′ cap comprising a Cap1 structure; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein:

-   -   (i) the Cap1 structure comprises         m7(3′OMeG)(5′)ppp(5′)(2′OMeA₁)pG₂, wherein A₁ is position +1 of         the RNA polynucleotide, and G₂ is position +2 of the RNA         polynucleotide; and     -   (ii) the cap proximal sequence comprises A₁ and G₂ of the Cap1         structure, and a sequence comprising: A₃A₄U₅ (SEQ ID NO: 5) at         positions +3, +4 and +5 respectively of the RNA polynucleotide.         13. The composition or medical preparation of any one of the         preceding embodiments, wherein the RNA polynucleotide comprises         a 5′ UTR and the cap proximal sequence is situated in a 5′ UTR.         14. A composition or medical preparation comprising a capped RNA         polynucleotide encoding a gene product, which RNA polynucleotide         comprises the formula:

wherein R¹ is CH₃, R² is OH or O—CH₃, and R³ is O—CH₃, wherein B₁ is any nucleobase, preferably A; B₂ is any nucleobase, preferably G; B₃ is any nucleobase, preferably A or C; B₄ is any nucleobase; and B₅ is any nucleobase, and wherein, when the RNA polynucleotide is administered to a subject, the levels of expression of the encoded gene product at about 6 hours after administration and at about 48 hours after administration do not differ by more than 5-fold. 15. The composition or medical preparation of embodiment 14, wherein, when the RNA polynucleotide is administered to a subject, the expression of the gene product is detectable at least 72 hours after administration. 16. The composition or medical preparation of embodiment 14 or 15, wherein:

the RNA polynucleotide comprises a 5′ UTR; and/or

the RNA polynucleotide further comprises a 3′ UTR sequence; and/or a polyA sequence.

17. The composition or medical preparation of any one of the preceding embodiments, wherein the RNA polynucleotide does not comprise a self-hybridizing sequence, optionally wherein a self-hybridizing sequence is a sequence that can hybridize to a sequence in a 5′ UTR or 3′ UTR of the RNA polynucleotide, e.g., wherein the self-hybridizing sequence is or comprises SEQ ID NO: 8. 18. The composition or medical preparation of any one of the preceding embodiments, wherein a 5′ UTR comprises a human alpha globin (hAg) 5′UTR or a fragment thereof, a TEV 5′ UTR or a fragment thereof, a HSP70 5′ UTR or a fragment thereof, or a c-Jun 5′ UTR or a fragment thereof. 19. The composition or medical preparation of any one of the preceding embodiments, wherein the 5′ UTR comprises a human alpha globin 5′ UTR provided in SEQ ID NO: 11, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto. 20. The composition or medical preparation of any one of embodiments 1 to 18, wherein the 5′ UTR comprises a human alpha globin 5′ UTR provided in SEQ ID NO: 12, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto. 21. The composition or medical preparation of any one of the preceding embodiments, wherein the 5′ UTR further comprises a T7 RNA polymerase promoter sequence. 22. The composition or medical preparation of any one of the preceding embodiments, wherein the 5′ cap structure is added co-transcriptionally or is not added enzymatically. 23. The composition or medical preparation of any one of the preceding embodiments, wherein the RNA polynucleotide comprises a 3′ UTR or a fragment thereof, optionally wherein the 3′ UTR sequence comprises SEQ ID NO: 13, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity thereto. 24. The composition or medical preparation of any one of embodiments 16-23, wherein the 3′ UTR or a proximal sequence thereto comprises a restriction site, optionally wherein the restriction site is a BamHI site or a XhoI site.

25. The composition or medical preparation of any one of the preceding embodiments, wherein a PolyA sequence comprises at least 100 nucleotides, optionally wherein:

(i) a polyA sequence is an interrupted sequence of adenine nucleotides; and/or

(ii) a polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.

26. The composition or medical preparation of embodiment 25, wherein a polyA sequence comprises the sequence of SEQ ID NO: 14. 27. The composition or medical preparation of any one of embodiments 16-26, wherein the 5′ cap, the 5′ UTR, the sequence encoding the payload, the 3′ UTR and the polyA sequence are situated disposed in a 5′ to 3′ orientation. 28. The composition or medical preparation of any one of the preceding embodiments, wherein the RNA polynucleotide comprises a Kozak sequence upstream of the sequence encoding a payload. 29. The composition or medical preparation of any one of the preceding embodiments, wherein the sequence encoding a payload comprises a promoter sequence and/or wherein the sequence encoding a payload comprises a sequence encoding a secretory signal peptide. 30. The composition or medical preparation of any one of the preceding embodiments, wherein an RNA polynucleotide comprises a sequence encoding a payload chosen from: a protein replacement polypeptide; an antibody agent; a cytokine; an antigenic polypeptide; a gene editing component; a regenerative medicine component or combinations thereof. 31. The composition or medical preparation of any one of the preceding embodiments, characterized in that, when assessed in an organism administered the composition or medical preparation comprising the RNA polynucleotide, elevated expression and/or increased duration of expression of the payload is observed relative to an appropriate reference comparator. 32. The composition or medical preparation of embodiment 30, wherein a payload is or comprises a protein replacement polypeptide, optionally wherein:

a protein replacement polypeptide comprises a polypeptide with aberrant expression in a disease or disorder;

a protein replacement polypeptide comprises an intracellular protein, an extracellular protein, or a transmembrane protein; and/or

a protein replacement polypeptide comprises an enzyme.

33. The composition or medical preparation of embodiment 32, wherein a disease or disorder with aberrant expression of a polypeptide includes but is not limited to: a rare disease, a metabolic disorder, a muscular dystrophy, a cardiovascular disease, or a monogenic disease. 34. The composition or medical preparation of embodiment 30, wherein a payload is or comprises an antibody agent, optionally wherein an antibody agent binds to a polypeptide expressed on a cell. 35. The composition or medical preparation of embodiment 34, wherein an antibody agent comprises a CD3 antibody, a Claudin 6 antibody, or a combination thereof. 36. The composition or medical preparation of embodiment 30, wherein a payload is or comprises a cytokine or a fragment or a variant thereof, optionally wherein a cytokine comprises: IL-12 or a fragment or variant or a fusion thereof, IL-15 or a fragment or a variant or a fusion thereof, GMCSF or a fragment or a variant thereof, or IFN-alpha or a fragment or a variant thereof. 37. The composition or medical preparation of embodiment 30, wherein a payload is or comprises an antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof, optionally wherein an antigenic polypeptide comprises one epitope from an antigen or a plurality of distinct epitopes from an antigen. 38. The composition or medical preparation of embodiment 37, wherein an antigenic polypeptide comprising a plurality of distinct epitopes from an antigen is polyepitopic. 39. The composition or medical preparation of embodiment 37 or 38, wherein an antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or a self-antigenic polypeptide. 40. The composition or medical preparation of embodiment 39, wherein a viral antigenic polypeptide comprises an HIV antigenic polypeptide, an influenza antigenic polypeptide, a Coronavirus antigenic polypeptide, a Rabies antigenic polypeptide, or a Zika virus antigenic polypeptide, optionally wherein a viral antigenic polypeptide is or comprises a Coronavirus antigenic polypeptide. 41. The composition or medical preparation of embodiment 40, wherein a Coronavirus antigen is or comprises a SARS-CoV-2 protein, optionally wherein a SARS-CoV-2 protein comprises a SARS-CoV-2 Spike (S) protein, or an immunogenic variant or an immunogenic fragment thereof. 42. The composition or medical preparation of embodiment 41, wherein the SARS-CoV-2 protein, or immunogenic variant or immunogenic fragment thereof, comprises proline residues at positions 986 and 987. 43. The composition or medical preparation of embodiment 41 or 42, wherein a SARS-CoV-2 S polypeptide:

(i) has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 9; or

(ii) is encoded by an RNA having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 10.

44. The composition or medical preparation of embodiment 30, wherein a payload is or comprises a tumor antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof, optionally wherein a tumor antigenic polypeptide comprises a tumor specific antigen, a tumor associated antigen, a tumor neoantigen, or a combination thereof. 45. The composition or medical preparation of embodiment 43 or 44, wherein a tumor antigenic polypeptide comprises p53, ART-4, BAGE, ss-catenin/m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan-A, MC1R, Myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Plac-1, Pm1/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE, WT, WT-1, or a combination thereof. 46. The composition or medical preparation of any one of embodiments 44-45, wherein a tumor antigenic polypeptide comprises a tumor antigen from a carcinoma, a sarcoma, a melanoma, a lymphoma, a leukemia, or a combination thereof. 47. The composition or medical preparation of embodiment 46, wherein a tumor antigenic polypeptide comprises: a melanoma tumor antigen; a prostate cancer antigen; a HPV16 positive head and neck cancer antigen; a breast cancer antigen; an ovarian cancer antigen; a lung cancer antigen, e.g., an NSCLC antigen. 48. The composition or medical preparation of embodiment 39, wherein a payload is or comprises a self-antigenic polypeptide or an immunogenic variant or an immunogenic fragment thereof, optionally wherein a self-antigenic polypeptide comprises an antigen that is typically expressed on cells and is recognized as a self-antigen by an immune system. 49. The composition or medical preparation of embodiment 48, wherein a self-antigenic polypeptide comprises: a multiple sclerosis antigenic polypeptide, a Rheumatoid arthritis antigenic polypeptide, a lupus antigenic polypeptide, a celiac disease antigenic polypeptide, a Sjogren's syndrome antigenic polypeptide, or an ankylosing spondylitis antigenic polypeptide, or a combination thereof. 50. The composition or medical preparation of any one of the preceding embodiments, wherein an RNA polynucleotide comprises a modified nucleoside in place of uridine, optionally wherein a modified nucleoside is selected from pseudouridine (Ψ), N1-methyl-pseudouridine (m1Ψ), and 5-methyl-uridine (m5U). 51. The composition or medical preparation of any one of the preceding embodiments, wherein an RNA polynucleotide further comprises one or more additional sequences, e.g., one or more additional payloads, and/or one or more regulatory elements. 52. The composition or medical preparation of any one of the preceding embodiments, wherein an RNA polynucleotide is characterized in that upon administration to an organism it increases translation efficiency of a payload, compared to administration of an otherwise similar RNA polynucleotide without a m7(3′OMeG)(5′)ppp(5′)(2′OMeA₁)pG₂ Cap. 53. The composition or medical preparation of any one of the preceding embodiments, wherein an RNA polynucleotide is characterized in that upon administration to an organism it increases the expression level and/or duration of expression of an encoded payload, compared to administration of an otherwise similar RNA polynucleotide without a m7(3′OMeG)(5′)ppp(5′)(2′OMeA₁)pG₂ cap, without a cap proximal sequence disclosed herein, and/or with a self-hybridizing sequence, optionally wherein:

(i) increased expression is determined at least 6 hours, at least 24 hours, at least 48 hours at least 72 hours, at least 96 hours or at least 120 hours after the administering, optionally wherein the increase in expression is at least 2-fold to 10-fold; or

(ii) elevated expression persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

54. The composition or medical preparation of any one of the preceding embodiments, wherein the RNA is formulated or is to be formulated as a liquid, a solid, or a combination thereof; wherein the RNA is formulated or is to be formulated for injection; or wherein the RNA is formulated or is to be formulated for intramuscular administration. 55. A pharmaceutical composition comprising an RNA polynucleotide of any one of the preceding embodiments, formulated as a particle, optionally wherein a particle is or comprises a lipid nanoparticle (LNP) or a lipoplex (LPX) particle. 56. The pharmaceutical composition of embodiment 55, wherein a lipid nanoparticle comprises each of: a cationic lipid; a sterol; a neutral lipid; and a lipid conjugate. 57. The pharmaceutical composition of embodiment 56, wherein the cationic lipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), the sterol is a cholesterol, the neutral lipid is or comprises a phospholipid, and the lipid conjugate is or comprises a polyethylene glycol (PEG)-lipid. 58. The pharmaceutical composition of embodiment 57, wherein the phospholipid is or comprises distearoylphosphatidylcholine (DSPC). 59. The pharmaceutical composition of embodiment 57, wherein the (PEG)-lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. 60. The pharmaceutical composition of any one of embodiments 56-58, wherein the lipid nanoparticle comprises each of: a. ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); b. a cholesterol; c. distearoylphosphatidylcholine (DSPC); and d. 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. 61. The pharmaceutical composition of any one of embodiments 56-60, wherein:

a neutral lipid is present in a concentration ranging from 5 to 15 mol percent of total lipids;

a cationically ionizable lipid is present in a concentration ranging from 40 to 55 mol percent of total lipids;

a steroid is present in a concentration ranging from 30 to 50 mol percent of total lipids; and/or

a pegylated lipid is present in a concentration ranging from 1 to 10 mol percent of total lipids.

62. The pharmaceutical composition of any one of embodiments 56-61, wherein a lipid nanoparticle comprise from 40 to 55 mol percent of a cationically ionizable lipid; from 5 to 15 mol percent of a neutral lipid; from 30 to 50 mol percent of a steroid; and from 1 to 10 mol percent of a pegylated lipid. 62. The pharmaceutical composition of embodiment 55, wherein an RNA lipoplex particle is obtainable by mixing an RNA polynucleotide with liposomes. 63. The composition or medical preparation of any one of embodiments 1-54, or a pharmaceutical composition of any one of embodiments 55-62, wherein the RNA is mRNA or saRNA. 64. The pharmaceutical composition of any one of embodiments 55-63, wherein a pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients. 65. The pharmaceutical composition of any one of embodiments 55-64, which is packaged as a kit, optionally wherein the kit further comprises instructions for use of said pharmaceutical composition for inducing an immune response in a subject. 66. A method of manufacturing a pharmaceutical composition of any one of embodiments 55-64, by combining an RNA polynucleotide with lipids to form lipid nanoparticles that encapsulate said RNA. 67. A nucleic acid template suitable to produce a cap1-capped RNA, in which the first five nucleotides transcribed from the template strand of the nucleic acid template comprise the sequence N₁pN₂pN₃pN₄pN₅, wherein N₁ is any nucleotide, preferably T; N₂ is any nucleotide, preferably C; N₃ is any nucleotide, preferably T or G; N₄ is any nucleotide; and N₅ is any nucleotide. 68. The nucleic acid template of embodiment 67, wherein the DNA template comprises: a 5′ UTR, a sequence encoding a payload, a 3′ UTR and a polyA sequence. 69. An in vitro transcription reaction comprising:

(i) a template DNA comprising a polynucleotide sequence complementary to an RNA polynucleotide sequence provided in any one of embodiments 1-54;

(ii) a polymerase (e.g., a T7 polymerase); and

(iii) an RNA polynucleotide.

70. The in vitro transcription reaction of embodiment 69, further comprising a 5′ cap or a 5′ cap analog, optionally wherein a 5′ cap or a 5′ cap analog is or comprises a Cap1 structure. 71. The in vitro transcription reaction of embodiment 69, wherein the RNA polynucleotide comprises a cap comprising a Cap1 structure; and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, optionally wherein the Cap1 structure comprises m7G(5′)ppp(5′)(2′OMeN₁)pN₂, wherein N₁ is position +1 of the RNA polynucleotide, and N₂ is position +2 of the RNA polynucleotide, and wherein N₁ and N₂ are each independently chosen from: A, C, G, or U. 72. An RNA polynucleotide isolated from an in vitro transcription reaction provided in any one of embodiments 69-71. 73. A method for producing a capped RNA comprising, transcribing a nucleic acid template in the presence of a cap structure, wherein the cap structure comprises G*ppp(m₁ ^(2-O))N₁pN₂, wherein N₁ is complementary to position +1 of the nucleic acid template and N₂ is complementary to position +2 of the nucleic acid template, and N₁ and N₂ are independently chosen from A, C, G or U, wherein position +3 of the nucleic acid template is any nucleotide, preferably T or G; position +4 of the nucleic acid template is any nucleotide; and position +5 of the nucleic acid template is any nucleotide, wherein G* comprises the following structure:

wherein

represents the bond by which G* is bound to the first phosphor atom of the ppp

group, R¹ is CH₃, R² is OH or O—CH₃, and R³ is O—CH₃. 74. A composition comprising a DNA polynucleotide comprising a sequence complementary to an RNA polynucleotide sequence provided in any one of embodiments 1-54. 75. The DNA polynucleotide composition of embodiment 74, which can be used to transcribe an RNA polynucleotide and/or, which is disposed in a vector. 76. A method comprising:

administering to a subject, a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle of any one of embodiments 55-64.

77. A method of inducing an immune response in a subject, comprising administering to a subject, a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle of any one of embodiments 55-64. 78. The method of embodiment 77, wherein an immune response is induced in a subject, optionally wherein an immune response is a prophylactic immune response or a therapeutic immune response. 79. A method of vaccination of a subject by administering a pharmaceutical composition comprising an RNA polynucleotide formulated in a lipid nanoparticle (LNP) or a lipoplex (LPX) particle of any one of embodiments 55-64. 80. The method of embodiment 79, vaccination generates an immune response, optionally wherein an immune response is a prophylactic immune response. 81. A method of decreasing interaction with IFIT1 of an RNA polynucleotide that comprises a 5′ cap and a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, the method comprising a step of:

providing a variant of the RNA polynucleotide that differs from the parental RNA polynucleotide by substitution of one or more residues within the cap proximal sequence, and

determining that interaction of the variant with IFIT1 is decreased relative to that of the parental RNA polynucleotide, optionally wherein the determining comprises administering the RNA polynucleotide or a composition comprising the same to a cell or an organism.

82. A method of producing a polypeptide comprising a step of:

providing an RNA polynucleotide that comprises a 5′ cap, a cap proximal sequence that comprises positions +1, +2, +3, +4, and +5 of the RNA polynucleotide, and a sequence encoding a payload;

wherein the RNA polynucleotide is characterized in that when assessed in an organism administered the RNA polynucleotide or a composition comprising the same, elevated expression and/or increased duration of expression of the payload is observed relative to an appropriate reference comparator.

83. A method of increasing translatability of an RNA polynucleotide that comprises a 5′ cap, a cap proximal sequence that comprises positions +1, +2, +3, +4, and +5 of the RNA polynucleotide and a sequence encoding a payload, the method comprising a step of:

providing a variant of the RNA polynucleotide that differs from a parental RNA polynucleotide by substitution of one or more residues within the cap proximal sequence; and

determining that expression of the variant is increased relative to that of the parental RNA polynucleotide, optionally wherein the determining comprises administering the RNA polynucleotide or a composition comprising the same to a cell or an organism.

84. The method of embodiment 83, wherein the increased translatability is assessed by increased expression and/or a persistence of expression of the payload. 85. The method of embodiment 84, wherein:

(i) increased expression is determined at least 6 hours, at least 24 hours, at least 48 hours at least 72 hours, at least 96 hours or at least 120 hours after the administering, optionally wherein the increase in expression is at least 2-fold to 10-fold; or

(ii) elevated expression persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

86. In a therapeutic RNA comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises:

including one or more of the following residues in a cap proximal sequence: A at position +1 of the RNA polynucleotide, G at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and U at position +5 of the RNA polynucleotide,

demonstrated to increase expression of the RNA when administered to a subject in an LNP formulation.

87. A method of increasing translation of an RNA polynucleotide comprising a 5′ cap that includes a Cap1 structure, a cap proximal sequence and a sequence encoding a payload, the improvement that comprises:

including one or more of the following residues in a cap proximal sequence: A at position +1 of the RNA polynucleotide, G at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and U at position +5 of the RNA polynucleotide.

88. The method of any one of embodiments 76-87, wherein one dose or a plurality of doses of a pharmaceutical composition is administered. 89. The method of any one of embodiments 76-88, wherein the method further comprises administration of one or more therapeutic agents. 90. The method of embodiment 89, wherein one or more therapeutic agents are administered before, after, or concurrently with administration of a pharmaceutical composition comprising an RNA polynucleotide. 91. The method of any one of embodiments 76-90, wherein the subject or organism is a mammal. 92. The method of embodiment 91, wherein the subject or organism is a human. 93. The method of any one of embodiments 76-93, wherein the subject has a disease or disorder disclosed herein. 94. A method of providing a framework for an RNA polynucleotide that comprises a 5′ cap, a cap proximal sequence, and a payload sequence, the method comprising a step of:

assessing at least two variants of the RNA polynucleotide, wherein:

each variant includes the same 5′ cap and payload sequence; and

the variants differ from one another at one or more specific residues of the cap proximal sequence;

wherein the assessing comprises determining expression levels and/or duration of expression of the payload sequence; and

selecting at least one combination of 5′ cap and a cap proximal sequence that displays elevated expression relative to at least one other combination.

95. The method of embodiment 94, wherein the assessing comprises administering the RNA construct or a composition comprising the same to a cell or an organism: 96. The method of embodiment 94 or 95, wherein:

the elevated expression is detected at a time point at least 6 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after the administering, optionally wherein the elevated expression is at least 2-fold to 10-fold; and/or

the elevated expression persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

97. The method of any one of embodiments 76-96, wherein the RNA polynucleotide comprises one or more features of an RNA polynucleotide provided in any one of embodiments 1-54. 98. The method of any one of embodiments 76-97, wherein the composition comprises a pharmaceutical composition of any one of embodiments 55-64.

EXEMPLIFICATION Example 1—Generation of an RNA Cassette that Provides Improved and Sustained Expression of the Encoded Protein

The present Example describes the generation of RNA cassettes with improved expression level of an encoded payload, and/or increased duration of expression of an encoded payload in vivo. The methods used in this Example are described below.

Methods:

mRNA Production

For templates, purified plasmids encoding codon-optimized murine erythropoietin (mEPO) were used. The plasmids corresponding to 5′ untranslated region sequences of tobacco etch virus 5′ leader RNA (TEV) or human α-globin (hAg) mRNA were linearized with BspQI (New England Biolabs, Cat #R0712L) to generate templates. The MEGAscript T7 RNA polymerase kit (Thermo Fisher Scientific, Cat #AMB1334-5) was used for transcription, and UTP was replaced with N1-methylpseudouridine (m1Ψ) 5′-triphosphate (TriLink, Cat #N-1081). Capping of the mRNAs was performed co-transcriptionally using anti-reverse Cap1 analogue CleanCap413 (TriLink, Cat #N-7413) at a final concentration of 3 mM. To obtain the desired transcripts generated with cap analogues, the initial GTP concentration in a transcription reaction was reduced from 7.5 mM to 1.5 mM and was incubated at 37° C. for 30 min in a hybridization chamber. The initial concentration of additional nucleotides including ATP, CTP and m1ΨP corresponded to the final 7.5 mM concentration. Adding of extra 1.5 mM GTP to the mixture was required after 30, 60, 90 and 120 minutes of incubation and incubated further at 37° C. for 30 minutes. The mRNAs were transcribed to contain 100 nt-long 3′ poly(A) tail (SEQ ID NO: 42). To remove the template 1/10 volume of DNA Turbo DNase (Thermo Fisher Scientific, Cat #AM1907) was added to the reaction mix and incubated the mixture at 37° C. for 15 minutes. The synthesized mRNA was isolated from the reaction mix by precipitation with half reaction volume of 8 M LiCl solution (Sigma-Aldrich, Cat #L7026). After chilling at −20° C. for at least 1 hour, the RNA pellet was collected by centrifuging at 17.000× g at 4° C. for 5 minutes. After washing the RNA pellet twice with at least 200 μl ice-cold 75% Ethanol solution, it was dissolved in nuclease free water. The concentration and quality of in vitro transcribed mRNA were measured on a NanoDrop2000C spectrophotometer (Thermo Fisher Scientific, Cat #ND-2000c). Aliquots of denatured IVT mRNAs were analyzed by electrophoresis in agarose gels containing 0.005% (v/v) GelRed™ nucleic acid gel stain (Masek T et al., (2005) Anal Biochem 336: 46-50). Small aliquots of mRNA samples were stored in siliconized tubes at −20° C. All mRNAs were cellulose-purified as described (Baiersdorfer M. et al. (2019) Mol Ther Nucleic Acids 15(15):26-35).

Mouse EPO-Specific Enzyme-Linked Immunosorbent Assay (ELISA)

For quantification of mouse EPO levels, plasma samples were collected from mice injected with IVT mRNA encoding for murine erythropoietin complexed with TransIT mRNA (Mirusbio, Cat #MIR2255) at the indicated time points and analyzed by mouse Erythropoietin DuoSet ELISA kit (R&D Systems, Cat #DY959). Flat-bottom 96-well plates were pre-coated with 2 μg/ml rat anti-mouse EPO capture antibody (100 μl/well) and incubated at room temperature (RT) overnight. The plates were washed three times with PBS containing 0.05% Tween-20 and incubated with 1% BSA (bovine serum albumin) (Sigma-Aldrich, Cat #2153) solution at RT for 2 hours to prevent non-specific binding of the antibody and washed again. A seven point standard curve using 2-fold serial dilutions and a high standard of 4000 pg/ml was applied. At a final volume of 50 μl plasma samples and standard diluted in 1% BSA solution were added to the appropriate wells and incubated at RT for 2 hours. After washing the plates, 100 μl of 1 μg/ml of rat biotinylated anti-mouse EPO detection antibody in 1% BSA solution was distributed to each well and incubated RT for 2 hours. The plates were washed and then incubated with 100 μl Streptavidin conjugated to horseradish peroxidase diluted (1:200) in 1% BSA solution at room temperature for 20 min. After washing, TMB 2-Component Microwell Peroxidase substrate solution (Medac Gmbh, Cat #50-76-11) was added to each well (100 μl/well). Samples were incubated at room temperature for 5 min, and 2 M sulfuric acid (R&D Systems, Cat #DY994) was added (50 l/well) to stop the reaction and absorbance was measured at 450 nm and 570 nm using an Infinite 200 Pro plate reader (Tecan).

Animal Protocol

All experiments were performed in accordance with federal policies on animal research using BALB/c female mice from Charles River Laboratories (Sandhofer, Germany) at an age of 6-12 weeks. For determining the translation of mRNA in vivo, 1-3 μg backbone- and nucleoside-modified cap1-TEV-mEPO mRNA or cap1-hAg-mEPO mRNA encoding for murine erythropoietin complexed with TransIT was injected intravenously into mice (3 mice/group). Blood was collected at 6, 24, 48, and 72 h after mRNA injection as described (Kariko K et al., (2012) Mol. Ther. 20:948-953; Mahin A J et al., (2016) Methods Mol Biol 1428:297-306) to avoid an impact of the sampling on the hematological parameters of the animals. In brief, blood (18 μL) was collected by puncture of the tail vein, mixed with 2 μL 0.2 M EDTA, and centrifuged in 20 μL Drummond microcaps glass microcapillary tubes (Sigma-Aldrich). After snapping the microcapillary tubes, the plasma was recovered for the measurement of plasma mEPO levels using the mEPO DuoSet ELISA Development kit (R&D Systems, Minneapolis, Minn., USA) and the Infinite 200 Pro plate reader (Tecan, Mannedorf, Switzerland). For details see EPO-ELISA description.

Results

This Example assesses the effect of sequence elements in an RNA on the expression level and/or duration of expression of a payload encoded by the RNA. The first evaluation focused on assessing the impact of self-hybridization sequences in 3′ UTR sequence of an RNA polynucleotide. RNA constructs with or without a self-hybridization sequence termed “Lig3” were generated and injected intravenously into mice. At 6, 24, 48, and 72 h after mRNA injection, blood was collected from the animals and assessed for expression of EPO—the polypeptide encoded by the RNA constructs.

As shown in FIG. 1, expression of EPO from constructs that did not have the Lig3 sequence was more robust. The expression level of EPO in these animals was higher than the control (RNA with Lig3) at 24 hours and 48 hours post-administration, and the duration of expression of EPO was also longer, with significant amounts of EPO detected up to 72 hours after administration. This data demonstrates that absence of self-hybridizing sequence in a 3′ UTR which can hybridize to other elements of the RNA, e.g., the 5′ UTR or the 3′ UTR itself as shown in FIG. 2, are beneficial for improving expression of an RNA encoded payload.

Next, additional sequence elements that could impact the expression of an RNA encoded payload were evaluated. One of the structural elements of an RNA which is required for translation and/or stability is a 5′ cap. The different 5′ Cap structures that can be used with an RNA are shown in FIGS. 3A-3H. It was previously shown that the IFIT1 (interferon-induced tetratricopeptidel) protein can bind to the 5′-end of the mRNA with very high affinity when the mRNA has a cap0 structure (PLoS pathogens (2013) 9, e1003663). The affinity of IFIT1's interaction with an mRNA reduces when it has a cap1 structure and this allows for preferential binding by translation factors such as the elongation factor eIF4E. Subsequently, it was also reported that the first 4 transcribed nucleotides at the 5′-end of the mRNA are located in a grove of the IFIT1 (PNAS (2017) 114(11): E2106-E2115). Based on these observations, it was hypothesized that the presence of a Cap1 structure on an RNA and the identity of the RNA sequence proximal to the 5′ Cap (also referred herein as “cap proximal sequence”) could impact the expression of a payload encoded by the RNA.

To test this hypothesis, RNAs having different residues in positions +3, +4 and +5 of the RNA sequence were generated and tested. Positions +1 and +2 of the RNA sequences tested were A and G respectively. Animals were injected intravenously with the various RNA constructs shown in FIG. 4. At 6, 24, 48, and 72 h after injection, blood was collected from the animals and assessed for expression of EPO—the polypeptide encoded by the RNA constructs.

EPO expression level at 6 hours and 24 hours after administration of RNA was comparable in all animals administered the various constructs. At 48 hours post-administration, some constructs resulted in better in vivo expression of EPO compared to others (compare AGAAU with AGACA at 48 hours). At 72 hours, the difference in in vivo EPO expression between animals dosed with the different constructs was even more significant with the AGACA construct showing more than a 10-fold reduced expression of EPO compared to several constructs including the AGAAU, AGAAC, AGCAA, AGCAC constructs.

This data demonstrates that the identity of the RNA sequences proximal to the 5′ cap has a significant impact on the in vivo expression level and/or duration of expression of an RNA encoded payload. In some embodiments, this data suggests that an RNA construct having an optimized cap proximal RNA sequence with increased payload expression and/or duration of expression in vivo, allows for administration of a lower dose of said RNA or a composition comprising the same to an organism relative to a comparator, e.g., an RNA sequence without an optimized cap proximal sequence.

Example 2: Exemplary Coronavirus Vaccine Constructs

The present Example describes certain coronavirus vaccine RNAs. Exemplified constructs include sequences encoding at least one epitope of a coronavirus spike protein, and various other structural elements and/or features. Among other things, documents that exemplified RNAs including, for example, a cap1 structure and proximal cap sequences as described herein are well expressed and strongly immunogenic.

TABLE 2 Exemplary sequences used in this example. Exemplary sequences SEQ ID NO: Description SEQUENCE Antigenic S protein sequences 17 S protein MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTN (amino acid) GTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKN NKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFS ALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDP LSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYN SASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFE LLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPC SFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECD IPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTM YICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKR SFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFG AGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNT LVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQ SKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRN FYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEID RLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFD EDDSEPVLKGVKLHYT 18 S protein auguuuguguuucuugugcugcugccucuugugucuucucagugugugaauuugacaacaagaacacagcugccaccagcuuauacaaauucuuuuaccag (CDS) aggaguguauuauccugauaaaguguuuagaucuucugugcugcacagcacacaggaccuguuucugccauuuuuuagcaaugugacaugguuucaugcaa uucaugugucuggaacaaauggaacaaaaagauuugauaauccugugcugccuuuuaaugauggaguguauuuugcuucaacagaaaagucaaauauuauu agaggauggauuuuuggaacaacacuggauucuaaaacacagucucugcugauugugaauaaugcaacaaauguggugauuaaagugugugaauuucaguu uuguaaugauccuuuucugggaguguauuaucacaaaaauaauaaaucuuggauggaaucugaauuuagaguguauuccucugcaaauaauuguacauuug aauaugugucucagccuuuucugauggaucuggaaggaaaacagggcaauuuuaaaaaucugagagaauuuguguuuaaaaauauugauggauauuuuaaa auuuauucuaaacacacaccaauuaauuuagugagagaucugccucagggauuuucugcucuggaaccucugguggaucugccaauuggcauuaauauuac aagauuucagacacugcuggcucugcacagaucuuaucugacaccuggagauucuucuucuggauggacagccggagcugcagcuuauuaugugggcuauc ugcagccaagaacauuucugcugaaauauaaugaaaauggaacaauuacagaugcuguggauugugcucuggauccucugucugaaacaaaauguacauuaa aaucuuuuacaguggaaaaaggcauuuaucagacaucuaauuuuagagugcagccaacagaaucuauugugagauuuccaaauauuacaaaucuguguccau uuggagaaguguuuaaugcaacaagauuugcaucuguguaugcauggaauagaaaaagaauuucuaauuguguggcugauuauucugugcuguauaauag ugcuucuuuuuccacauuuaaauguuauggagugucuccaacaaaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuugugaucagaggu gaugaagugagacagauugcccccggacagacaggaaaaauugcugauuacaauuacaaacugccugaugauuuuacaggaugugugauugcuuggaauuc uaauaauuuagauucuaaagugggaggaaauuacaauuaucuguacagacuguuuagaaaaucaaaucugaaaccuuuugaaagagauauuucaacagaaau uuaucaggcuggaucaacaccuuguaauggaguggaaggauuuaauuguuauuuuccauuacagagcuauggauuucagccaaccaauggugugggauauc agccauauagagugguggugcugucuuuugaacugcugcaugcaccugcaacaguguguggaccuaaaaaaucuacaaauuuagugaaaaauaaaugugug aauuuuaauuuuaauggauuaacaggaacaggagugcugacagaaucuaauaaaaaauuucugccuuuucagcaguuuggcagagauauugcagauaccaca gaugcagugagagauccucagacauuagaaauucuggauauuacaccuuguucuuuugggggugugucugugauuacaccuggaacaaauacaucuaauca gguggcugugcuguaucaggaugugaauuguacagaagugccaguggcaauucaugcagaucagcugacaccaacauggagaguguauucuacaggaucua auguguuucagacaagagcaggaugucugauuggagcagaacaugugaauaauucuuaugaaugugauauuccaauuggagcaggcauuugugcaucuuau cagacacagacaaauuccccaaggagagcaagaucuguggcaucucagucuauuauugcauacaccaugucucugggagcagaaaauucuguggcauauucu aauaauucuauugcuauuccaacaaauuuuaccauuucugugacaacagaaauuuuaccugugucuaugacaaaaacaucuguggauuguaccauguacauu uguggagauucuacagaauguucuaaucugcugcugcaguauggaucuuuuuguacacagcugaauagagcuuuaacaggaauugcuguggaacaggauaa aaauacacaggaaguguuugcucaggugaaacagauuuacaaaacaccaccaauuaaagauuuuggaggauuuaauuuuagccagauucugccugauccuuc uaaaccuucuaaaagaucuuuuauugaagaucugcuguuuaauaaagugacacuggcagaugcaggauuuauuaaacaguauggagauugccugggugaua uugcugcaagagaucugauuugugcucagaaauuuaauggacugacagugcugccuccucugcugacagaugaaaugauugcucaguacacaucugcuuua cuggcuggaacaauuacaagcggauggacauuuggagcuggagcugcucugcagauuccuuuugcaaugcagauggcuuacagauuuaauggaauuggagu gacacagaauguguuauaugaaaaucagaaacugauugcaaaucaguuuaauucugcaauuggcaaaauucaggauucucugucuucuacagcuucugcuc ugggaaaacugcaggauguggugaaucagaaugcacaggcacugaauacucuggugaaacagcugucuagcaauuuuggggcaauuucuucugugcugaau gauauucugucuagacuggauaaaguggaagcugaagugcagauugauagacugaucacaggaagacugcagucucugcagacuuaugugacacagcagcu gauuagagcugcugaaauuagagcuucugcuaaucuggcugcuacaaaaaugucugaaugugugcugggacagucaaaaagaguggauuuuuguggaaaag gauaucaucugaugucuuuuccacagucugcuccacauggagugguguuuuuacaugugacauaugugccagcacaggaaaagaauuuuaccacagcacca gcaauuugucaugauggaaaagcacauuuuccaagagaaggaguguuugugucuaauggaacacauugguuugugacacagagaaauuuuuaugaaccuca gauuauuacaacagauaauacauuugugucaggaaauugugauguggugauuggaauugugaauaauacaguguaugauccacugcagccagaacuggauu cuuuuaaagaagaacuggauaaauauuuuaaaaaucacacaucuccugauguggauuuaggagauauuucuggaaucaaugcaucuguggugaauauucag aaagaaauugauagacugaaugaaguggccaaaaaucugaaugaaucucugauugaucugcaggaacuuggaaaauaugaacaguacauuaaauggccuugg uacauuuggcuuggauuuauugcaggauuaauugcaauugugauggugacaauuauguuauguuguaugacaucauguuguucuuguuuaaaaggaugu uguucuuguggaagcuguuguaaauuugaugaagaugauucugaaccuguguuaaaaggagugaaauugcauuacaca 19 S protein RBD MFVFLVLLPLVSSQCVVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSP (amino TKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF acid)(V05) RKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPK 20 S protein RBD auguuuguguuucuugugcugcugccucuugugucuucucaguguguggugagauuuccaaauauuacaaaucuguguccauuuggagaaguguuuaau (CDS) (V05) gcaacaagauuugcaucuguguaugcauggaauagaaaaagaauuucuaauuguguggcugauuauucugugcuguauaauagugcuucuuuuuccacau uuaaauguuauggagugucuccaacaaaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuugugaucagaggugaugaagugagacagau ugcccccggacagacaggaaaaauugcugauuacaauuacaaacugccugaugauuuuacaggaugugugauugcuuggaauucuaauaauuuagauucua aagugggaggaaauuacaauuaucuguacagacuguuuagaaaaucaaaucugaaaccuuuugaaagagauauuucaacagaaauuuaucaggcuggaucaa caccuuguaauggaguggaaggauuuaauuguuauuuuccauuacagagcuauggauuucagccaaccaauggugugggauaucagccauauagaguggug gugcugucuuuugaacugcugcaugcaccugcaacaguguguggaccuaaa 21 S protein RBD/ MFVFLVLLPLVSSQCVVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSP Fibritin TKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLF (amino acid) RKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKGS (V05) PGSGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPG 22 S protein RBD/ auguuuguguuucuugugcugcugccucuugugucuucucaguguguggugagauuuccaaauauuacaaaucuguguccauuuggagaaguguuuaau Fibritin gcaacaagauuugcaucuguguaugcauggaauagaaaaagaauuucuaauuguguggcugauuauucugugcuguauaauagugcuucuuuuuccacau (CDS) (V05) uuaaauguuauggagugucuccaacaaaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuugugaucagaggugaugaagugagacagau ugcccccggacagacaggaaaaauugcugauuacaauuacaaacugccugaugauuuuacaggaugugugauugcuuggaauucuaauaauuuagauucua aagugggaggaaauuacaauuaucuguacagacuguuuagaaaaucaaaucugaaaccuuuugaaagagauauuucaacagaaauuuaucaggcuggaucaa caccuuguaauggaguggaaggauuuaauuguuauuuuccauuacagagcuauggauuucagccaaccaauggugugggauaucagccauauagaguggug gugcugucuuuugaacugcugcaugcaccugcaacaguguguggaccuaaaggcucccccggcuccggcuccggaucugguuauauuccugaagcuccaag agaugggcaagcuuacguucguaaagauggcgaauggguauuacuuucuaccuuuuuaggccggucccuggaggugcuguuccagggccccggc  9 S protein PP MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTN (amino acid) GTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKN (V08/V09) NKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFS ALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDP LSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYN SASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFE LLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPC SFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECD IPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTM YICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKR SFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFG AGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNT LVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQ SKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRN FYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEID RLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFD EDDSEPVLKGVKLHYT 23 S protein PP auguuuguguuucuugugcugcugccucuugugucuucucagugugugaauuugacaacaagaacacagcugccaccagcuuauacaaauucuuuuaccag (CDS) (V08) aggaguguauuauccugauaaaguguuuagaucuucugugcugcacagcacacaggaccuguuucugccauuuuuuagcaaugugacaugguuucaugcaa uucaugugucuggaacaaauggaacaaaaagauuugauaauccugugcugccuuuuaaugauggaguguauuuugcuucaacagaaaagucaaauauuauu agaggauggauuuuuggaacaacacuggauucuaaaacacagucucugcugauugugaauaaugcaacaaauguggugauuaaagugugugaauuucaguu uuguaaugauccuuuucugggaguguauuaucacaaaaauaauaaaucuuggauggaaucugaauuuagaguguauuccucugcaaauaauuguacauuug aauaugugucucagccuuuucugauggaucuggaaggaaaacagggcaauuuuaaaaaucugagagaauuuguguuuaaaaauauugauggauauuuuaaa auuuauucuaaacacacaccaauuaauuuagugagagaucugccucagggauuuucugcucuggaaccucugguggaucugccaauuggcauuaauauuac aagauuucagacacugcuggcucugcacagaucuuaucugacaccuggagauucuucuucuggauggacagccggagcugcagcuuauuaugugggcuauc ugcagccaagaacauuucugcugaaauauaaugaaaauggaacaauuacagaugcuguggauugugcucuggauccucugucugaaacaaaauguacauuaa aaucuuuuacaguggaaaaaggcauuuaucagacaucuaauuuuagagugcagccaacagaaucuauugugagauuuccaaauauuacaaaucuguguccau uuggagaaguguuuaaugcaacaagauuugcaucuguguaugcauggaauagaaaaagaauuucuaauuguguggcugauuauucugugcuguauaauag ugcuucuuuuuccacauuuaaauguuauggagugucuccaacaaaauuaaaugauuuauguuuuacaaauguguaugcugauucuuuugugaucagaggu gaugaagugagacagauugcccccggacagacaggaaaaauugcugauuacaauuacaaacugccugaugauuuuacaggaugugugauugcuuggaauuc uaauaauuuagauucuaaagugggaggaaauuacaauuaucuguacagacuguuuagaaaaucaaaucugaaaccuuuugaaagagauauuucaacagaaau uuaucaggcuggaucaacaccuuguaauggaguggaaggauuuaauuguuauuuuccauuacagagcuauggauuucagccaaccaauggugugggauauc agccauauagagugguggugcugucuuuugaacugcugcaugcaccugcaacaguguguggaccuaaaaaaucuacaaauuuagugaaaaauaaaugugug aauuuuaauuuuaauggauuaacaggaacaggagugcugacagaaucuaauaaaaaauuucugccuuuucagcaguuuggcagagauauugcagauaccaca gaugcagugagagauccucagacauuagaaauucuggauauuacaccuuguucuuuugggggugugucugugauuacaccuggaacaaauacaucuaauca gguggcugugcuguaucaggaugugaauuguacagaagugccaguggcaauucaugcagaucagcugacaccaacauggagaguguauucuacaggaucua auguguuucagacaagagcaggaugucugauuggagcagaacaugugaauaauucuuaugaaugugauauuccaauuggagcaggcauuugugcaucuuau cagacacagacaaauuccccaaggagagcaagaucuguggcaucucagucuauuauugcauacaccaugucucugggagcagaaaauucuguggcauauucu aauaauucuauugcuauuccaacaaauuuuaccauuucugugacaacagaaauuuuaccugugucuaugacaaaaacaucuguggauuguaccauguacauu uguggagauucuacagaauguucuaaucugcugcugcaguauggaucuuuuuguacacagcugaauagagcuuuaacaggaauugcuguggaacaggauaa aaauacacaggaaguguuugcucaggugaaacagauuuacaaaacaccaccaauuaaagauuuuggaggauuuaauuuuagccagauucugccugauccuuc uaaaccuucuaaaagaucuuuuauugaagaucugcuguuuaauaaagugacacuggcagaugcaggauuuauuaaacaguauggagauugccugggugaua uugcugcaagagaucugauuugugcucagaaauuuaauggacugacagugcugccuccucugcugacagaugaaaugauugcucaguacacaucugcuuua cuggcuggaacaauuacaagcggauggacauuuggagcuggagcugcucugcagauuccuuuugcaaugcagauggcuuacagauuuaauggaauuggagu gacacagaauguguuauaugaaaaucagaaacugauugcaaaucaguuuaauucugcaauuggcaaaauucaggauucucugucuucuacagcuucugcuc ugggaaaacugcaggauguggugaaucagaaugcacaggcacugaauacucuggugaaacagcugucuagcaauuuuggggcaauuucuucugugcugaau gauauucugucuagacuggauccuccugaagcugaagugcagauugauagacugaucacaggaagacugcagucucugcagacuuaugugacacagcagcu gauuagagcugcugaaauuagagcuucugcuaaucuggcugcuacaaaaaugucugaaugugugcugggacagucaaaaagaguggauuuuuguggaaaag gauaucaucugaugucuuuuccacagucugcuccacauggagugguguuuuuacaugugacauaugugccagcacaggaaaagaauuuuaccacagcacca gcaauuugucaugauggaaaagcacauuuuccaagagaaggaguguuugugucuaauggaacacauugguuugugacacagagaaauuuuuaugaaccuca gauuauuacaacagauaauacauuugugucaggaaauugugauguggugauuggaauugugaauaauacaguguaugauccacugcagccagaacuggauu cuuuuaaagaagaacuggauaaauauuuuaaaaaucacacaucuccugauguggauuuaggagauauuucuggaaucaaugcaucuguggugaauauucag aaagaaauugauagacugaaugaaguggccaaaaaucugaaugaaucucugauugaucugcaggaacuuggaaaauaugaacaguacauuaaauggccuugg uacauuuggcuuggauuuauugcaggauuaauugcaauugugauggugacaauuauguuauguuguaugacaucauguuguucuuguuuaaaaggaugu uguucuuguggaagcuguuguaaauuugaugaagaugauucugaaccuguguuaaaaggagugaaauugcauuacaca 24 S protein PP auguucguguuccuggugcugcugccucugguguccagccagugugugaaccugaccaccagaacacagcugccuccagccuacaccaacagcuuuaccaga (CDS) (V09) ggcguguacuaccccgacaagguguucagauccagcgugcugcacucuacccaggaccuguuccugccuuucuucagcaacgugaccugguuccacgccauc cacguguccggcaccaauggcaccaagagauucgacaaccccgugcugcccuucaacgacgggguguacuuugccagcaccgagaaguccaacaucaucagag gcuggaucuucggcaccacacuggacagcaagacccagagccugcugaucgugaacaacgccaccaacguggucaucaaagugugcgaguuccaguucugca acgaccccuuccugggcgucuacuaccacaagaacaacaagagcuggauggaaagcgaguuccggguguacagcagcgccaacaacugcaccuucgaguacg ugucccagccuuuccugauggaccuggaaggcaagcagggcaacuucaagaaccugcgcgaguucguguuuaagaacaucgacggcuacuucaagaucuaca gcaagcacaccccuaucaaccucgugcgggaucugccucagggcuucucugcucuggaaccccugguggaucugcccaucggcaucaacaucacccgguuuc agacacugcuggcccugcacagaagcuaccugacaccuggcgauagcagcagcggauggacagcuggugccgccgcuuacuaugugggcuaccugcagccua gaaccuuccugcugaaguacaacgagaacggcaccaucaccgacgccguggauugugcucuggauccucugagcgagacaaagugcacccugaaguccuuca ccguggaaaagggcaucuaccagaccagcaacuuccgggugcagcccaccgaauccaucgugcgguuccccaauaucaccaaucugugccccuucggcgagg uguucaaugccaccagauucgccucuguguacgccuggaaccggaagcggaucagcaauugcguggccgacuacuccgugcuguacaacuccgccagcuuca gcaccuucaagugcuacggcguguccccuaccaagcugaacgaccugugcuucacaaacguguacgccgacagcuucgugauccggggagaugaagugcggc agauugccccuggacagacaggcaagaucgccgacuacaacuacaagcugcccgacgacuucaccggcugugugauugccuggaacagcaacaaccuggacuc caaagucggcggcaacuacaauuaccuguaccggcuguuccggaaguccaaucugaagcccuucgagcgggacaucuccaccgagaucuaucaggccggcag caccccuuguaacmcguggaaggcuucaacugcuacuucccacugcaguccuacggcuuucagcccacaaauggcgugggcuaucagcccuacagaguggu ggugcugagcuucgaacugcugcaugccccugccacagugugcggcccuaagaaaagcaccaaucucgugaagaacaaaugcgugaacuucaacuucaacgg ccugaccggcaccggcgugcugacagagagcaacaagaaguuccugccauuccagcaguuuggccgggauaucgccgauaccacagacgccguuagagaucc ccagacacuggaaauccuggacaucaccccuugcagcuucggcggagugucugugaucaccccuggcaccaacaccagcaaucagguggcagugcuguacca ggacgugaacuguaccgaagugcccguggccauucacgccgaucagcugacaccuacauggcggguguacuccaccggcagcaauguguuucagaccagagc cggcugucugaucggagccgagcacgugaacaauagcuacgagugcgacauccccaucggcgcuggaaucugcgccagcuaccagacacagacaaacagcccu cggagagccagaagcguggccagccagagcaucauugccuacacaaugucucugggcgccgagaacagcguggccuacuccaacaacucuaucgcuaucccca ccaacuucaccaucagcgugaccacagagauccugccuguguccaugaccaagaccagcguggacugcaccauguacaucugcggcgauuccaccgagugcu ccaaccugcugcugcaguacggcagcuucugcacccagcugaauagagcccugacagggaucgccguggaacaggacaagaacacccaagagguguucgccc aagugaagcagaucuacaagaccccuccuaucaaggacuucggcggcuucaauuucagccagauucugcccgauccuagcaagcccagcaagcggagcuuca ucgaggaccugcuguucaacaaagugacacuggccgacgccggcuucaucaagcaguauggcgauugucugggcgacauugccgccagggaucugauuugc gcccagaaguuuaacggacugacagugcugccuccucugcugaccgaugagaugaucgcccaguacacaucugcccugcuggccggcacaaucacaagcggc uggacauuuggagcaggcgccgcucugcagauccccuuugcuaugcagauggccuaccgguucaacggcaucggagugacccagaaugugcuguacgagaa ccagaagcugaucgccaaccaguucaacagcgccaucggcaagauccaggacagccugagcagcacagcaagcgcccugggaaagcugcaggacguggucaac cagaaugcccaggcacugaacacccuggucaagcagcuguccuccaacuucggcgccaucagcucugugcugaacgauauccugagcagacuggacccuccu gaggccgaggugcagaucgacagacugaucacaggcagacugcagagccuccagacauacgugacccagcagcugaucagagccgccgagauuagagccucu gccaaucuggccgccaccaagaugucugagugugugcugggccagagcaagagaguggacuuuugcggcaagggcuaccaccugaugagcuucccucaguc ugccccucacggcgugguguuucugcacgugacauaugugcccgcucaagagaagaauuucaccaccgcuccagccaucugccacgacggcaaagcccacuu uccuagagaaggcguguucguguccaacggcacccauugguucgugacacagcggaacuucuacgagccccagaucaucaccaccgacaacaccuucguguc uggcaacugcgacgucgugaucggcauugugaacaauaccguguacgacccucugcagcccgagcuggacagcuucaaagaggaacuggacaaguacuuuaa gaaccacacaagccccgacguggaccugggcgauaucagcggaaucaaugccagcgucgugaacauccagaaagagaucgaccggcugaacgagguggccaa gaaucugaacgagagccugaucgaccugcaagaacuggggaaguacgagcaguacaucaaguggcccugguacaucuggcugggcuuuaucgccggacuga uugccaucgugauggucacaaucaugcuguguugcaugaccagcugcuguagcugccugaagggcuguuguagcuguggcagcugcugcaaguucgacga ggacgauucugagcccgugcugaagggcgugaaacugcacuacaca Foldon 25 Foldon (amino GSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPG acid) 26 Foldon (CDS) ggaucugguuauauuccugaagcuccaagagaugggcaagcuuacguucguaaagauggcgaauggguauuacuuucuaccuuuuuaggccggucccugga ggugcuguuccagggccccggc 5′-UTR (hAg-Kozak) 27 5'-UTR AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC 3′-UTR (FI element) 13 3'-UTR CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACC UCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCC AAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAA CCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGC CACACC A30L70 14 A30L70 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

Primary pharmacodynamics studies were performed in BALB/c mice to test the immunogenicity of the vaccine candidates shown in the following table.

TABLE 3 Vaccine candidates mRNA Vaccine type Vaccine encoded antigen BNT162a1 uRNA RBD (Receptor Binding Domain) of SARS-CoV-2 Spike protein (S protein) BNT162b1 modRNA RBD (Receptor Binding Domain) of SARS-CoV-2 Spike protein (S protein) BNT162b2 modRNA Modified version of SARS-CoV-2 Spike protein (S protein) BNT162c1 saRNA RBD (Receptor Binding Domain) of SARS-CoV-2 Spike protein (S protein) In the study, four groups of each eight female BALB/c mice were immunized once with the animal trial material at three different doses, or with buffer (control group; see Table 4). While the clinical trial material will be diluted in saline, the animal trial material was diluted in PBS including 300 mM sucrose. As this is the storage buffer of the material itself, the test items are representative for the vaccine that will be used in the planned clinical trials. Immunizations were given IM using a dose volume of 20 μL.

TABLE 4 Study design Dose Blood End of Group No of Vaccine Immunization volume collection in-life No animals dose Day [μL]/route Day phase 1 8 buffer 0 20/IM 7, 14, 21 28 2 8 Low 0 20/IM 7, 14, 21 28 3 8 Medium 0 20/IM 7, 14, 21 28 4 8 High 0 20/IM 7, 14, 21 28

Blood of immunized animals was collected on days 7, 14, 21 and 28, and analyzed for the antibody immune response by ELISA and pseudovirus-based neutralization assay (pVNT).

SARS-CoV-2-S specific antibody responses directed against the recombinant S1 subunit or the RBD were detected by ELISA. In brief, high protein-binding 96-well plates (MaxiSorp ELISA plates, VWR International GmbH, Cat. No. 7341284) were coated with 100 ng recombinant S1 subunit (Sino Biological Inc., Cat. No. 40591-V08H) or RBD (Sino Biological Inc., Cat. No. 40592-V02H) per well in 100 μL coating buffer (50 mM sodium carbonate-bicarbonate buffer, pH9.6) overnight at 4° C. Plates were washed three times with 300 μL/well 1× phosphate-buffered saline (PBS, VWR International GmbH, Cat. No. 0780-10L) supplemented with 0.01% Tween 20 (Carl Roth GmbH & Co. KG, Cat. No. 9127.1) and blocked with 250 μL/well 1× Casein Blocking Buffer (Sigma-Aldrich GmbH, Cat No. B6429-500 m1) for 1 hour at 37° C. on a microplate shaker. Plates were again washed three times with 300 μL/well 1×PBS supplemented with 0.01% Tween 20 and incubated with mouse serum samples diluted in 1× Casein Blocking Buffer for 1 hour at 37° C. on a microplate shaker. Plates were washed three times with 300 μL/well 1×PBS supplemented with 0.01% Tween 20 and subsequently incubated with Peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Ltd., Cat. No. 115-036-071; diluted 1:7500 in 1× Casein Blocking Buffer) for 45 minutes at 37° C. on a microplate shaker. Plates were washed three times with 300 μL/well 1×PBS supplemented with 0.01% Tween 20 and 100 μL/well TMB substrate (Biotrend Chemiekalien GmbH, Cat. No. 4380A) was added. Plates were incubated for 8 min at room temperature and the reaction stopped by addition of 100 μL 25% sulphuric acid (VWR International GmbH, Cat. No. 1007161000). Plates were read on a microplate reader and the recorded absorbance at 450 nm corrected by subtracting the reference absorbance at 620 nM.

Functional antibody responses to the vaccine candidates were detected by pVNT. The pVNT uses a replication-deficient vesicular stomatitis virus (VSV) that lacks the genetic information for the VSV envelope glycoprotein G but contains an open-reading frame (ORF) for green fluorescent protein (GFP). VSV/SARS-CoV-2 pseudovirus was generated according to a published protocol (Hoffmann et al., Cell, 2020; PMID 32142651). The pseudotype virus bears the SARS-CoV-2 S protein, which mediates cell entry. Therefore, the pseudovirus can be inactivated by neutralizing antibodies that bind SARS-CoV-2 S. This inactivation can be analyzed via in vitro methods.

In brief, 4×10⁴ Vero 76 cells (ATCC® CRL-1587™) per well were seeded in a 96-well plate (Greiner Bio-One GmbH, Cat. No. 655160) in 150 μL/well DMEM (Thermo Fisher Scientific, Cat. No. 61965059) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich GmbH, Cat. No. F7524). Cells were incubated for 4 to 6 hours at 37° C. and 7.5% CO₂. Meanwhile, mouse serum samples were diluted 1:6 up to 1:768 in DMEM/10% FBS in two-fold dilution steps. Diluted serum samples were combined with an equal volume of titrated and pre-diluted VSV/SARS CoV-2 pseudovirus supernatant, resulting in a serum dilution ranging from 1:12 up to 1:1536. The pseudovirus/serum dilution mix was incubated for 5 min at RT on a microplate shaker at 750 rpm with an additional 5 min incubation at RT without agitation. 50 μL/well pseudovirus/serum dilution mix was added to the seeded Vero-76 cells with the applied pseudovirus volume per well corresponding to 200 infectious units (IU). Each dilution of serum samples was tested in duplicate wells. Cells were incubated for 16 to 24 hours at 37° C. and 7.5% CO₂. Vero 76 cells incubated with pseudovirus in the absence of mouse sera were used as positive controls. Vero 76 cells incubated without pseudovirus were used as negative controls. After the incubation, the cell culture plates were removed from the incubator, placed in an IncuCyte Live Cell Analysis system (Essen Bioscience) and incubated for 30 min prior to the analysis. Whole well scanning for brightfield and GFP fluorescence was performed using a 4× objective. To calculate the neutralizing titer, infected GFP-positive cell number per well was compared with the pseudovirus positive control. Mean values of the pseudovirus positive control multiplied by 0.5 represent the pseudovirus neutralization 50% (pVN50). Serum samples with mean values below this cut-off exhibit >50% virus neutralization activity, respectively.

Immunogenicity Study of BNT162a1 (RBL063.3)

To dissect the potency of the LNP-formulated uRNA vaccine coding for BNT162a1, BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine will be investigated by focusing on the antibody immune response.

ELISA data 7, 14, 21 and 28 d after the first immunization show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 5).

Immunogenicity Study of BNT162b1 (RBP020.3)

To dissect the potency of the LNP-formulated modRNA vaccine coding for BNT162b1, BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine will be investigated by focusing on the antibody immune response.

ELISA data 7, 14, 21 and 28 d after the first immunization show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 6). Sera obtained 14, 21, and 28 d after immunization show high SARS-CoV-2 pseudovirus neutralization, especially sera from mice immunized with 1 or 5 μg BNT162b1 and correlating with the strong increase of IgG antibody titers (FIG. 7).

Immunogenicity Study of BNT162c1 (RBS004.3)

To dissect the potency of the LNP-formulated saRNA vaccine coding for BNT162c1, BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine will be investigated by focusing on the antibody immune response.

ELISA data 7, 14 and 21 d after the first immunization show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 8). Sera obtained 14, and 21 d after immunization show dose-dependent SARS-CoV-2 pseudovirus neutralization activity (FIG. 9).

Immunogenicity Study of LNP-Formulated uRNA Encoding the Viral S Protein-V8 (SEQ ID NO: 7, 8) (RBL063.1)

To dissect the potency of the LNP-formulated uRNA vaccine coding for the viral S protein-V8 (RBL063.1), BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine will be investigated by focusing on the antibody immune response.

ELISA data 7, 14, 21 and 28 d after the first immunization are available that show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 10). Sera obtained 14, 21 and 28 d after immunization show dose-dependent SARS-CoV-2 pseudovirus neutralization activity (FIG. 11).

Immunogenicity Study of BNT162b2 (RBP020.1)

To dissect the potency of the vaccine BNT162b2 (RBP020.1), the immunogenicity of the construct was investigated. For this purpose, a dose titration study in BALB/c mice was initiated where the immune response will be analyzed focusing on the antibody immune response.

ELISA data 7, 14, and 21 d after the first immunization are available that show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 12). Sera obtained 14, and 21 d after immunization show dose-dependent SARS-CoV-2 pseudovirus neutralization activity (FIG. 13).

Immunogenicity Study of the LNP-Formulated saRNA Encoding the Viral S Protein-V9 (SEQ ID NO: 7, 9) (RBS004.2)

To dissect the potency of the LNP-formulated saRNA vaccine coding for V9, BALB/c mice were immunized IM once as outlined in Table 3. The immunogenicity of the RNA vaccine will be investigated by focusing on the antibody immune response.

ELISA data 7, 14, and 21 d after the first immunization are available that show an early, dose-dependent immune activation against the S1 protein and the receptor binding domain (FIG. 14). Sera obtained 14, and 21 d after immunization show dose-dependent SARS-CoV-2 pseudovirus neutralization activity (FIG. 15).

The above data demonstrate an immune response for both the RBD with a trimerization domain (“V5”) and the mutated full-length S protein (“V8”/“V9”) in vivo in all tested platforms (including the vaccines BNT162a1, BNT162b1, BNT162b2, and BNT162c1). The antibody immune response was already seen at very early time points by ELISA (i.e., at 7 d post-immunization) Importantly, induced antibodies were able to efficiently neutralize SARS-COV-2 pseudovirus infection in vitro. Also, the induction of an antibody response using a very low immunization dose of 0.2 μg/mouse when using the modRNA platform (BNT162b1, BNT162b2) as well as the saRNA platform (BNT162c1) indicates a high potency of the vaccine candidates.

In mice, BNT162b2 induced a higher antigen-specific titer compared to BNT162b1 encoded with the identical RNA platform. As expected, the immunogenicity in mice against the antigens differs between the RNA platforms. In mice, the most immunogenic platform based on antigen-specific antibody induction is the modRNA followed by saRNA. The uRNA platform induces the lowest antigen-specific antibody titer.

Example 3: LNP Formulation

An exemplary LNP delivery system was developed to effectively and safely deliver therapeutic nucleic acids into the cytosol of various cell types after local administration in vivo. The early formulation work was performed with several promising LNP formulations and surrogate RNA coding for luciferase. The aim of the experiments was to correlate the effect of different ionizable cationic lipids on the efficacy of RNA delivery by LNPs in vivo. Formulations were compared in terms of RNA encapsulation efficiency, apparent pKa, LNP size and polydispersity.

Among the screened cationic lipids, ALC-0315 exhibited suitable physical characteristics regarding particle size, homogeneity, and RNA encapsulation efficiency.

Based on this the ALC-0315/DSPC/CHOL/ALC-0159 prototype was submitted for in vivo screening. The results presented in FIG. 16 summarize the in vivo testing of two independent pilot batches using luciferase (Luc) RNA. The results demonstrate improved potency of the ALC-0315 prototype as compared to an internal benchmark (ALC-0218). On the basis of these studies, ALC-0315 was identified as a highly potent cationic lipid and brought forward for further product development studies.

The formulation screening procedure described above involves intravenous administration resulting in delivery primarily to the liver. The mechanism of LNP uptake into hepatocytes is driven by binding of endogenous apolipoproteins to the LNP followed by receptor-mediated endocytosis e.g. through low density lipoprotein receptors. In order to investigate whether the same mechanism is involved for an intramuscular administration, Luc RNA containing LNPs comprising ALC-0315 were injected intravenously (0.3 mg/kg) and intramuscularly (0.2 mg/kg) into ApoE knockout mice in the presence or absence of recombinant human ApoE3. As control, wild-type C57B1/6 mice were also treated by the different routes of administration. RNA-LNP were pre-incubated with recombinant human ApoE3 (1 mg encapsulated mRNA with 1 mg ApoE3) for 1 hour at room temperature (RT) prior to administration. Luc expression was monitored at 4, 24, 72 and 96 hours post administration (FIG. 17).

When mice were administered intravenously, Luc expression was detected in the wild-type C57B1/6 mice. In the ApoE knockout mice Luc expression was significantly reduced however when preincubated with exogenous ApoE the expression of Luc was recovered to similar expression levels as wild-type mice (FIG. 18).

In vivo Luc expression experiments using mouse models showed, that similar mechanisms are involved in the uptake of RNA-LNP in case of intramuscular administration as for intravenous administration, and this is not only true for hepatocytes but also for the cells local to the administration site.

In vivo experiments after intramuscular administration of the final ALC-0315/DSPC/CHOL/ALC-0159, confirmed minimal drainage with regards to biodistribution, immunogenicity (vaccine activity) and tolerability.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Further, it should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the claims that follow. 

1.-147. (canceled)
 148. A composition or medical preparation comprising an RNA polynucleotide comprising: a 5′ cap comprising a Cap1 structure; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein: (i) the Cap1 structure comprises m7(3′OMeG)(5′)ppp(5′)(2′OMeA₁)pG₂, wherein A₁ is position +1 of the RNA polynucleotide, and G₂ is position +2 of the RNA polynucleotide; and (ii) the cap proximal sequence comprises A₁ and G₂ of the Cap1 structure, and a sequence comprising: A₃A₄U₅ (SEQ ID NO: 5) at positions +3, +4 and +5 respectively of the RNA polynucleotide.
 149. The composition or medical preparation of claim 148, wherein the RNA polynucleotide comprises a 5′ UTR and the cap proximal sequence is situated in a 5′ UTR.
 150. The composition or medical preparation of claim 149, wherein the 5′ UTR comprises a human alpha globin (hAg) 5′ UTR or a fragment thereof.
 151. The composition or medical preparation of claim 150, wherein the 5′ UTR further comprises a Kozak sequence.
 152. The composition or medical preparation of claim 151, wherein the 5′ UTR is or comprises the nucleotide sequence of SEQ ID NO:
 27. 153. The composition or medical preparation of claim 148, wherein the RNA polynucleotide further comprises a 3′ UTR sequence.
 154. The composition or medical preparation of claim 153, wherein the 3′ UTR sequence comprises the nucleotide sequence of SEQ ID NO:
 13. 155. The composition or medical preparation of claim 148, wherein the RNA polynucleotide further comprises a polyA sequence.
 156. The composition or medical preparation of claim 155, wherein the polyA sequence comprises at least 100 nucleotides.
 157. The composition or medical preparation of claim 156, wherein: (i) the polyA sequence is an interrupted sequence of adenine nucleotides; and/or (ii) the polyA sequence comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.
 158. The composition or medical preparation of claim 157, wherein the polyA sequence comprises the nucleotide sequence of SEQ ID NO:
 14. 159. The composition or medical preparation of claim 148, wherein the RNA polynucleotide comprises, in a 5′ to 3′ orientation, the 5′ cap, the cap proximal sequence, a 5′ UTR, the sequence encoding a payload, a 3′ UTR, and a polyA sequence.
 160. The composition or medical preparation of claim 148, wherein the RNA polynucleotide comprises a modified nucleoside in place of uridine.
 161. The composition or medical preparation of claim 160, wherein the modified nucleoside is or comprises N1-methyl-pseudouridine (m₁ψ).
 162. The composition or medical preparation of claim 148, wherein the sequence encoding a payload is or comprises a sequence encoding a protein replacement polypeptide; a sequence encoding an antibody agent; a sequence encoding a cytokine; a sequence encoding an antigenic polypeptide; a sequence encoding gene editing component; a sequence encoding a regenerative medicine component, or combinations thereof.
 163. The composition or medical preparation of claim 162, wherein the sequence encoding a payload is or comprises a sequence encoding an antigenic polypeptide.
 164. The composition or medical preparation of claim 163, wherein the antigenic polypeptide comprises an epitope from an antigen.
 165. The composition or medical preparation of claim 163, wherein the antigenic polypeptide comprises a plurality of distinct epitopes from one or more antigens.
 166. The composition or medical preparation of claim 165, wherein the antigenic polypeptide comprising a plurality of distinct epitopes from one or more antigens is a polyepitopic polypeptide.
 167. The composition or medical preparation of claim 163, wherein the antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or a self-antigenic polypeptide.
 168. The composition or medical preparation of claim 148, wherein the RNA polynucleotide is or comprises mRNA.
 169. A pharmaceutical composition comprising an RNA polynucleotide formulated as a particle, wherein the RNA polynucleotide comprises: a 5′ cap comprising a Cap1 structure; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein: (i) the Cap1 structure comprises m7(3′OMeG)(5′)ppp(5′)(2′OMeA₁)pG₂, wherein A₁ is position +1 of the RNA polynucleotide, and G₂ is position +2 of the RNA polynucleotide; and (ii) the cap proximal sequence comprises A₁ and G₂ of the Cap1 structure, and a sequence comprising: A₃A₄U₅ (SEQ ID NO: 5) at positions +3, +4 and +5 respectively of the RNA polynucleotide.
 170. The pharmaceutical composition of claim 169, wherein the particle is or comprises a lipid nanoparticle (LNP) or a lipoplex (LPX) particle.
 171. The pharmaceutical composition of claim 170, wherein the particle is or comprises a LNP comprising a cationically ionizable lipid; a sterol; a phospholipid; and a pegylated lipid.
 172. The pharmaceutical composition of claim 171, wherein the concentration of the cationically ionizable lipid in the pharmaceutical composition is within a range of about 40 to about 55 mole percent, the concentration of the sterol in the pharmaceutical composition is within a range of about 30 to about 50 mole percent, the concentration of the phospholipid in the pharmaceutical composition is within a range of about 5 to about 15 mole percent, and the concentration of the pegylated lipid in the pharmaceutical composition is within a range of about 1 to about 10 mole percent.
 173. The pharmaceutical composition of claim 172, wherein the cationically ionizable lipid is or comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), the sterol is or comprises cholesterol, the phospholipid is or comprises distearoylphosphatidylcholine, and the pegylated lipid is or comprises (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide).
 174. The pharmaceutical composition of claim 173, wherein the concentration of ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) in the pharmaceutical composition is within a range of about 40 to about 55 mole percent, the concentration of cholesterol in the pharmaceutical composition is within a range of about 30 to about 50 mole percent, the concentration of distearoylphosphatidylcholine (DSPC) in the pharmaceutical composition is within a range of about 5 to about 15 mole percent, and the concentration of 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide in the pharmaceutical composition is within a range of about 1 to about 10 mole percent.
 175. The pharmaceutical composition of claim 169, which is formulated as a liquid.
 176. The pharmaceutical composition of claim 169, which is formulated as a solid.
 177. A composition or medical preparation comprising an RNA polynucleotide comprising: a 5′ cap comprising a Cap1 structure; a cap proximal sequence comprising positions +1, +2, +3, +4, and +5 of the RNA polynucleotide; and a sequence encoding a payload, wherein: (i) the Cap1 structure comprises m7(3′OMeG)(5′)ppp(5′)(2′OMeA₁)pG₂, wherein A₁ is position +1 of the RNA polynucleotide, and G₂ is position +2 of the RNA polynucleotide; and (ii) the cap proximal sequence comprises A₁ and G₂ of the Cap1 structure, and a sequence comprising: A₃N₄N₅ at positions +3, +4 and +5 respectively of the RNA polynucleotide, wherein N₄ and N₅ are each independently selected from A, G, C, and U. 